BIOCHIMIE, 1978, 60, 365-379.
Comparative study of kinetoplast DNA in cuhure, blood and intracellular forms of Trypanosoma cruzi. (27-2-1978).
* Unitd de Pharmacologie Moldculaire, lnstitut Gustave Roussy, 94800 Villejui[, France. *" Biological Laboratory, University of Kent, Canterbury, Kent, England.
R6sum6.
Summary.
Une 6tude c o m p a r a t i v e du DNA kin6toplastiq u e (kDNA) de ,, f o r m e 1 ,, a 6t6 entreprise sur T r y p a n o s o m a cruz/ & diff~rents s t a d e s de son cycle 6volutif (formes de culture 6pimastigotes, s a n g u i n e s t r y p o m a s t i g o t e s et intracellulaires amastiqotes). Le kDNA de ces t r y p a n o s o m e s p o s s 6 d e des propri6t6s c o m m u n e s (densit6 de flottation 1,698-9 g / c m 3 - - coefficient de s6dim e n t a t i o n $20, w ~ 5500 - - f o r m e m o l 6 c u l a i r e en r 6 s e a u c o m p l e x e de minicercles). Plusieurs diff6rences ont 6t6 not6es. I1 y a proportionnellem e a t p l u s d e kDNA darts les t r y p a n o s o m e s de f o r m e s a n g u i n e ce qui s u q g 6 r e q u e leur kin6top l a s t e r e n f e r m e plusieurs r ~ s e a u x de kDNA p u i s q u e la taille de ces r 6 s e a u x est identique darts les trois formes. Ces r6sultats sont corrobor6s p a r l ' a r r a n g e m e n t ultrastructural du kDNA de la f o r m e s a n g u i n e en 4 d o u b l e couches, diff6rent de celui d e s f o r m e s de culture et intracellulaire en u n e seule d o u b l e couche. Q u e l q u e s g r a n d e s m o l 6 c u l e s circulaires h~t6rog 6 n e s en taille ont 6t6 6 g a l e m e n t o b s e r v 6 e s d a n s les formes s a n q u i n e s et intracellulaires. Apr~s digestion du k D N A p a r les e n d o n u c l e a ses de restriction, les trac6s 61ectrophor6tiques m o n t r e n t q u e l q u e s diff6rences d'ordre quantitatif entre les 3 formes de T. cruz/.
A c o m p a r a l i v e s t u d y h a s b e e n m a d e on the k i n e t o p l a s t DNA (kDNA) form I in culture epimastigote, blood t r y p o m a s t i q o t e a n d intracellular a m a s t i g o t e s t a g e s of T r y p a n o s o m a cruz/. The b a s i c properties of the kDNA in all 3 forms w e r e identical. Thus the DNA w a s in the form of n e t w o r k s of density 1.698-9 g / c m 3 a n d with s e d i m e n t a t i o n coefficients (S20w) of ~ 5500, the n e t w o r k s b e i n g c o m p o s e d of l a r g e c o m p l e x e s of minicircular a n d a p p a r e n t l y linear molecules, the former h a v i n g contour lengths of 0.45 ~m. S e v e r a l differences w e r e noted, The ultrastructural a r r a n g e m e n t of the kDNA in the kinetoplast of the blood s t a g e consisted of three to four double rows of DNA a s c o m p a r e d to one d o u b l e l a y e r e d r o w in the other two stages. There w a s p r o p o r t i o n a t e l y m o r e kDNA in the b l o o d s t a g e s , s u g g e s t i n g that, since the netw o r k s h a v e a p p a r e n t l y the s a m e size (see above), m o r e t h a n one is present, DNA loops situated at the p e r i p h e r y of the kDNA networks w e r e o b s e r v e d in higher proportion in blood a n d intracellular forms. Dimeric a n d oligomeric circles w e r e p r e s e n t in the kDNA of the blood a n d intracellular s t a g e s in m u c h g r e a t e r proportion t h a n in culture e p i m a s t i g o t e s t a g e s . Few l a r g e circular molecules, h e t e r o g e n e o u s in size, w e r e also o b s e r v e d in intracellular b l o o d s t a g e s . There w e r e s o m e differences, m a i n l y quantitative, in the gel electrophoresis p a t t e r n s after e n d o n u c l e a s e digestion.
Introduction.
the base of the flagellum. Electron microscopy has shown that the kinetoplast is a part of the single mitochondrion of these organisms. In the species investigated so far, the kinetoplast has been found to contain DNA which can be distinguished from that in the nucleus by its buoyant density in CsC1 gradients. Kinetoplast DNA (kDNA) can be regar-
Guy F. RIOU ~" and Wins¢on E. GUTTERIDGE +*
A characteristic feature of trypanosomes is the presence of a structure termed the kinetoplast near
To whom all correspondence should be addressed.
366
G. F. R i o u and W . E. Gulteridge.
d e d as the e q u i w a l e n t of m i t o c h o n d r i a l DNA of o t h e r e u k a r y o t i c celIs. R e v i e w s of its p r o p e r t i e s a n d p o s s i b l e f u n c t i o n s h a v e b e e n p u b l i s h e d [1-4]. A c h a r a c t e r i s t i c f e a t u r e of Trltpanosoma cruzi w h i c h d i s t i n g u i s h e s it f r o m A f r i c a n t r y p a n o s o m e s , is t h a t g r o w t h a n d d i v i s i o n in ~he m a m m a l o c c u r i n t r a c e l l u l a r l y , u s u a l l y in t h e m u s c l e t i s s u e of t h e h e a r t a n d , a l i m e n t a r y t r a c t . T h o u g h it is n o w gen e r a l l y a g r e e d t h a t t h e s e i n t r a c e l l u l a r stages h a v e s h o r t f r e e flagella, t h e y a r e r e f e r r e d to as a m a s t i g a t e stages. M o r p h o l o g i c a l l y , t h e y a r e q u i t e distinct from the trypomastigote forms which occur in t h e b l o o d a n d w h i c h , al,though u n a b l e to d i v i d e , s e r v e as a v e h i c u l e b o t h f o r s p r e a d i n g t h e i n f e c t i o n a r o u n d t h e hos~ a n d also f o r i n f e s t i n g the insect vector during feeding. Growth and division in t h e v e c t o r o c c u r m a i n l y as e p i m a s t i g o t e s ; f o r m s s i m i l a r to t h e l a t t e r c a n r e a d i l y be g r o w n in c u l t u r e (see r e f e r e n c e 5 for r e v i e w of t h e biol o g y of t h e o r g a n i s m ) . E a r l i e r w o r k in V i l l e j u i f sho"wed t h a t the cult u r e e p i m a s t i g o t e f o r m s of T. cruzi c o n t a i n a v e r y h i g h p r o p o r t i o n of k D N A (20-25 p e r c e n t of t h e total cell DNA) [6]. It w a s also s h o w n t h a t t h i s k D N A w a s a p p a r e n t l y in t h e f o r m of an a s s o c i a t i o n of l i n e a r m o l e c u l e s a n d m i n i c i r c u l a r ,molecules ( c o n t o u r l e n g t h 0.45 i~m) w h i c h t h u s f o r m e d a c o m p l e x n e t w o r k [1, 7], T h e r e a r e 20-30,000 of t h e m i n i c i r c u l a r m o l e c u l e s p e r k i n e t o p l a s t [81. T h e i r r e p l i c a t i o n ,appeared to be as d e s c r i b e d b y C a i r n s f o r the c h r o m o s o m e s of Escherichia colt [9], It is not k n o w n w h a t g e n e t i c i n f o r m a t i o n t h e y contain. A s i m i l a r o r g a n i s a t i o n of k D N A w a s l a t e r f o u n d in oth, er s p e c i e s of t r y p ~ n o s o m e s ( r e v i e w e d i n ref. 2), t h o u g h m o s t r e c e n t s t u d i e s [10, 11, r e v i e w e d i n 4] suggest t h a t at least in s o m e s p e c i e s of Crithidia a n d Trgpanosoma, t h e l i n e a r k D N A m o l e c u l e s i~n f a c t o c c u r as m a x i c i r c l e s ( f r o m 6 to 11 ~ m c o n t o u r l e n g t h d e p e n d i n g on s p e c i e s ) . It has b e e n s u g g e s t e d t h a t t h e s e r e p r e s e n t t h e t r u e m i t o c h o n d r i a l D N A of t h e s e o r g a n i s m s . T h e r e is g o o d e v i d e n c e t h a t t h e b l o o d t r y p o m a s t i g o t e stages of T. cruzi, in c o n t r a s t to t h e b l o o d stages of A f r i c a n t r y p a n o s o m e s , c a t a b o l i s e g l u c o s e in l a r g e p a r t to GO2 via a T,CA c y c l e a n d respiratory chain ; the mitochondri,a are cristate, TCA c y c l e s e n z y m e s a r e p r e s e n t a n d e y t o c h r o m e s h a v e b e e n d e s c r i b e d [ r e v i e w e d in 5]. I t is l i k e l y t h e r e f o r e t h a t w h a t e v e r g e n e t i c i n f o r m a t i o n is c o n t a i n e d w i t h i n t h e k ] ) N A it is :expressed in t h e s e stages. F u r t h e r m o r e , p r e l i m i : n a r y u l t r a s t r u c t u r a l s t u d i e s in ¥ i l l e j u i f a.nd in o t h e r l a b o r a t o r i e s [12, 13] of b l o o d t r y p o m a s t i g o t e f o r m s s h o w e d t h a t t h e
BIOCH1M1E, 1978, 60, n ° 4.
o r g a n i s a t i o n of t h e DN,A in t h e kinetopl,ast w a s q u i t e d i s t i n c t f r o m t h a t in t h e c u l t u r e f o r m s . At this t i m e , m e t h o d s h a d b e e n d e v e l o p e d in C a n t e r b u r y to i s o l a t e b l o o d t r y p o m a s t i g o t e a n d i n t r a e e l l u l a r a m a s t i g o t e stages f r o m i n f e c t e d r a i s [14]. It w a s d e c i d e d , t h e r e f o r e , to c a r r y out a j o i n t i n v e s t i g a t i o n of t h e k D N A of t h e c u l t u r e e p i m a s t i g o t e , blood trypomastigote and intracellular amastigote f o r m s of T. cruzi. A p r e l i m i n a r y a c c o u n t of s o m e of t h e s e r e s u l t s has b e e n p r e s e n t e d [15].
Materials
and
Methods.
Organism. The Sonya strain of Trgpanosoma cruzi was used, except where there is a clear indication to the contrary. Most of the experiments involving restriction endonucleases were carried out after it had been cloned (Cover and Gutteridge, in preparation). The strain was maintained in Porton-derived XL mice by syringe passage (subcutaneous inoculation) at intervals of 2-3 weeks. Parasites for experiments were obtained from rats (wt range 100-120 g) which had been subjected ,~ 14, days earlier to whole body ~-irradiation (580 rads) prior to inoculation sub-cutaneously with 107 blood trypomastigotes from mice [14]. Culture epimastigote forms were grown at 28°C in a modified LIT m e d i u m [16]. In a few experiments, clearly indicated, culture forms of the Tehuantepec strain [17] were also used. When labelled DNA was required, 5 ~ C i / m l of [methyl-3H]-thymidine (specific activity 20: Ci/mM) was added to medium at the beginning of the culture period. Isolation of blood stages. The method used [14] was based on the DEAE cellulose column method described by Lanham and Godfrey [18]. It involved the use of their phosphate saline glucose buffer at a pH value of 7.5 and at an ionie strength of 0.2~)6 and was carried out at room temperature. The m a j o r i t y of the trypanosomes were eluted withou t retardation as a m a j o r peak. No contamination with red cells, white cells or platelets could he observed by light microscopy. As judged by light microscopy, organisms were motile and normal in form ; they were. infective to mice. Isolation of intracellular stages. Isolation was made from rat skeletal muscle by a p r o c e d u r e described elsewhere [14]. This involves disruption of the muscle tissue in an MSE tissue homogeniser, filtration through gauze and then incubation of the homogenate with deoxyribonuclease, collagenase and trypsin to destroy muscle cell nuclei, break down gelatin-like proteins and degrade muscle cell debris to small particles. A pellet fraction is th~en obtained by differential centrifugation contaminated only by a few white (1-2 per cent) and red (2-11) per cent) cells. In most preparations, these blood cells were removed by centrifugation in a linear sucrose gradient (0.25.-0.70 M). Between 1 and 2 per cent of the parasites thus isolated are epimastigotes and trypomastigotes ; the remainder are amastigotes. Parasites isolated by this method are apparently undamaged as judged by light and electron microscopy and are metabolically active at least as far as respiration and nucleic acid and protein syn-
kDNA
o[ T. cruzi.
t h e s i s are concerned. The i n f e c t i v i t y for mice of b o t h purified p r e p a r a t i o n s a n d i n i t i a l cell h o m o g e n a t e s ean be a c c o u n t e d for by t h e e p i m a s t i g o t e s a n d t r y p o m a s t i gores p r e s e n t in t h e m .
Electron microscopy. P r e p a r a t i o n s were fixed w i t h g l u t a r a l d e h y ~ e - o s m i u m t e t r a o x i d e a n d e m b e d d e d in Epon. T h i n sections w e r e d o u b l e - s t a i n e d w i t h u r a n y l acetate a n d lead citrate as described p r e v i o u s l y [19].
Extraction of total cell DNA. T h i s w a s e x t r a c t e d by a m o d i f i c a t i o n of t h e m e t h o d of M a r m u r [20, 21]. T r y p a n o z o m e s , a f t e r l y s i s in s a r k o s y l (final c o n c e n t r a t i o n 1 per cent w / v ) i n SSC (NaC1, 0.15 M ; Nas citrate, 0.015 M, pH 7.4) were digested w i t h p r o n a s e (13 grade, Calbioehem, 1 m g / m l ) for 2 h r at 37~C (the stock solut i o n of t h e e n z y m e (10 m g / m l ) w a s self-digested for 2 h r at 37°C in SSC). The digest w a s t h e n e x t r a c t e d once w i t h c h l o r o f o r m / i s o a m y l alcohol (24:1 by vol). The a q u e o u s phase was dialysed extensively against SSC a n d t h e n i n c u b a t e d a t 37°C, first w i t h RNase A (pre-treat.ed by h e a t i n g to 90°C for 11) rain) at a conc e n t r a t i o n o f 100 ,~g/ml for ~ hr, t h e n w i t h RNase TI (10 U / m l ) a n d finally w i t h p r o n a s e , T h e DNA w a s e x t r a c t e d a g a i n w i t h organic s o l v e n t s as above a n d red i a l y s e d a g a i n s t 0.1 S~SC, Fraciionalion of kDNA. Total DNA e x t r a c t s were f r a e t i o n a t e d b y u l t r a e e n t r i f u g a t i o n in a p r o p i d i u m diiodide-(PErI)-CsC1 g r a d i e n t (PI)I 51~)igxg/ml ; 50 Ti R o t o r ; 24 h r ; 20'°C; 40,(~00 r p m ) [22]. T h e lower b a n d w h i c h c o n t a i n s kDNA in its c o n s t r a i n e d f o r m was collected a n d t h e PDI w a s e x t r a c t e d w i t h i s o p r o p a n o l [23]. The kl)NA w a s freed of r e s i d u a l n u c l e a r DN~A by s u b s e q u e n t u l t r a e e n t r i f u g a t i o n in a H'g÷+ --Cs2SO, g r a d i e n t [6], The kDNA f o r m I w a s u s e d in e x p e r i m e n t s described in t h i s s t u d y ,
367
w a s l a y e r e d on distilled w a t e r a s a h y p o p h a s e a t 4°C. DNA f r o m P I ~ p h a g e (9,90~ n u c l e o t i d e s pairs, 6.4 X 1~6 d a l t o n s ) w a s u s e d as i n t e r n a l size s t a n d a r d .
A n a l y t i c a l ultraeentri[uyation. B u o y a n t d e n s i t y e x p e r i m e n t s were p e r f o r m e d at 25°C for 24 h r at 44,770 r p m i n a B e c k m a n m o d e l E c e n t r i f u g e . The b u o y a n t d e n s i t y g r a d i e n t of V i n o g r a d a n d H e a r s t [24] w a s u s e d in t h e e a I e u l a t i o n of t h e r e s u l t s w i t h reference to E. colt DNA (density 0 = 1.710 g/eroS). T h e DNA m a r k e r w a s 2C phage DNA of Bacillus subtilis (0 = 1.742 g/em3). T h e s e d i m e n t a t i o n coefficients of kDNA were m e a s u r e d by b a n d s e d i m e n t a t i o n a n a l y s i s i n 3 M CsC1 0.01 M Nas-EDTA (pH 8.0) according to t h e m e t h o d of V i n o g r a d et al [25] except t h a t a lower speed of r o t a t i o n w a s u s e d (4000 r p m at 20°C in a B e c k m a n ANJ rotor). Each cell w a s filled w i t h 15 lal of the DNA s o l u t i o n p r e v i o u s l y d i a l y s e d a g a i n s t 2 M NaCl-O.O,1 M Na~-EDrfA. R e s t r i c t i o n endonucleases cleavaye of kDNA. Several r e s t r i c t i o n e n z y m e s were used to e x a m i n e t h e overall c o m p o s i t i o n of kDNA. Eco RI e n d o n u c l e a s e w a s p u r i fied e s s e n t i a l l y according to Y o s h i m o r i [26]. H i n d II A-Ill a n d Hae IH e n d o n u c l e a s e s were respectively purified b y t h e m e t h o d s of S m i t h a n d Wilcox [27] a n d Middleton et al. [28] Hpa II e n d o n u e l e a s e , p r e p a red as described b y S h a r p e t a l [29], w a s k i n d l y provided b y P. N a r d e u x . T a q I p r e p a r e d b y t h e m e t h o d described by Sato et al. [30] w a s a g e n e r o u s gift of Dr. J. F e u n t e u n . T h e activities of l h e v a r i o u s re'strietion e n z y m e s were checked u n d e r c o n d i t i o n s described in the c o r r e s p o n d i n g references, u s i n g ~ DNA a n d SV 40 DNA. T h e DNA f r a g m e n t s of kDNA were s e p a r a t e d by e l e c t r o p h o r e s i s in vertical 2 per cent a g a r o s e slab gels as described by D a n n a et al. [31]. After m i g r a t i o n ,
F16. 1. - - Photomicrographs of thin sections of the S o n y a strain of T. cruzi s h o w i n y detail of the kinctoplast. A, eultfire e p i m a s t i g o t e ; B, blood t r y p o m a s t i g o t e ; C, i n t r a e e l l u l a r anaastigote.
Preparation ol kDNA for electron microscopy, kI>NA m o l e c u l e s w e r e e x a m i n e d u n d e r a P h i l i p s EM 300 elect r o n microscope a f t e r being s p r e a d w i t h o u t f o r m a m i d [1] or w i t h f o r m a m i d as described p r e v i o u s l y [7] h u t w i t h t h e following modifications. 55 ~I of DNA solut i o n in SSC were m i x e d w i t h ¢5 lxl of f o r m a m i d (Fluka) a n d 15 ]xl of c y t o c h r o m e c (1 m g / m l ) (Calbioehem). T h e m i x t u r e was m a i n t a i n e d at 4°C. T h e DNA BIOCHIMIE, 1978, 60, n ° 4,
t h e y were located by e t h i d i u m b r o m i d e s t a i n i n g a n d p h o t o g r a p h e d u n d e r UV l i g h t w i t h a red filter. W h e n labelled DNA w a s used, t h e r a d i o a c t i v i t y of each b a n d w a s d e t e r m i n e d a f t e r slicing t h e gel into 1 r a m t h i c k slices. The slices were dissolved o v e r n i g h t in 1 ml of Soluene 350 (Packard) at 37°C. S c i n t i l l a t i o n f l u i d w a s added a n d slices were e s t i m a t e d for r a d i o a c t i v i t y in a liquid s c i n t i l l a t i o n s p e c t r o m e t e r .
G. F. R i o u a n d W . E. G u t t e r i d g e .
368
Ultracentrifugation in CsCl.
Results. K inetoplast ultrastructure. E x a m i n a t i o n of thin sections of the 3 forms of T. cruzi c o n f i r m e d results d e s c r i b e d p r e v i o u s l y about the u l t r a s t r u c t u r a l c o n f o r m a t i o n of kDNA
Culture
U l t r a c e n t r i f u g a t i o n in i s o p y c n i c CsC1 gradients s h o w e d the p r e s e n c e in all 3 f o r m s of 2 DNA components : a n u c l e a r c o m p o n e n t of density 1.710 g / c m 3 ( ~ 51 per c e n t G + C) and a rapidb a n d i n g k i n e t o p l a s t c o m p o n e n t of density 1.6981.699 g / c m 3 (-~ 40 per cent G + C) (fig. 2). The
Intracellular
Blood f o r m
form
form
1.698
1.710
1
1.742
l
1.710
1.742 1.742 1.698 1.698 1.710 FI6. 2. - - Isopycnic ultracentrifugation analysis of DNA from T. eruzi. Phage 2 G DNA from Bacillus subtilis was used as a marker (5 ---- 1.7'42 g/cm3. 9 ---- 1.710 g/era3 was nuclear DNA. ~ = 1.69,8 g/cm3 was kDNA).
in isolated culture epimastigote [19] and b l o o d t r y p o m a s t i g o t e [123 forms and the s t r u c t u r e of the kinetoplast in i n t r a c e l l u l a r ,amastigote stages e x a m i n e d in situ [13, 32, 33]. In the culture forms, the kinetoplast is localized at the base of the flagellum adjac.ent to the basal body. It is b o u n d to m i t 0 c h o n d r i a l m e m b r a n e s . The kDNA appears as a double r o w of regular and w e l l d e f i n e d elect r o n dense m a t e r i a l (fig. 1A). In the blood forms the kinetoplast is globular and the kDNA is arranged in 3 or 4 double l a y e r e d r o w s of fibers (fig. 1B). In the i n t r a c e l l u l a r forms several k i n d s of figures are obserwed for the k i n e t o p l a s t and its DNA. The most frequent, N 70 p e r cent of thin sections observed, show a s t r u c t u r e of kDNA s i m i l a r to that of the e p i m a s t i g o t e forms, t h o u g h w i t h a less o r d e r e d fibrillar a r r a n g e m e n t (fig. 1C). The kDNA found in other figures is a r r a n g e d in several double r o w s and appears to c o r r e s p o n d to an i n t e r m e d i a t e state b e t w e e n the t r y p o m a s t i g o t e and the epimastigote forms.
BIOCH1MIE, 1978, 60, n ° 4.
results of calculations f r o m the u l t r a c e n t r i f u g a t i o n data about the p r o p o r t i o n s of kDNA in the differ e n t forms of T. cruzi are given in table I. As
FIG. 3. - - Band sedimentation analysis of kDNA from the blood forms of T. eruzi in 3 M CsCl at !tOO0 rpm, Photographs were taken at 2 rain intervals.
k D N A of T . c r u z i . e x p e c t e d f r o m e l e c t r o n m i c r o g r a p h s of u l t r a t h i n s e c t i o n s , t h e r e is p r o p o r t i o n a t e l y m o r e k D N A in t h e b l o o d stages t h a n in t h e t~ther 2 stages.
369
3 stages. It is in t h e f o r m of a c o m p l e x n e t w o r k of m a n y t h o u s a n d s of m i n i c i r c u l a r m o l e c u l e s and a p p a r e n t l o n g l i n e a r p i e c e s ( w h i c h m a y b e p a r t of
TABLE I.
Properties of kDNA in the different forms of T. cruzi. Form of T. cruzi.
Culture Blood Intraeellular
kDNA as per cent of total DNA
20.3 ~ 0.60 3 3 . 6 -~- 4.1 2 6 . 0 ~__ 2 . 0
(4) (7) (4)
Sedimentation coefficients of kDNA networks (in S)
Size ol kDNA networks (l~m'-') •
3650 -t- 200 (3) 3730 ~- 380 (3) 4510 and 4550
121 ~__ 18 (13) 125 ~__ 26 (17) 128 -4- 23 (17)
Values for the percentage of kDNA were derived from densitometer tracings obtained after analytical ultraeentrifugation in CsC1 gradients of total DNA as described in figure 2. To calculate their size the kDNA networks were spread without formamid as described in [1]. Electron mierographs were all taken with the same magnification in order to abtain the entire network in the centre of the plate. Numbers in brackets are numbers of experiments done or networks measured. The sedimentation coefficients and the sizes of the kDNA networks were determined from k D N A form I as described in Materials and Methods.
T h e o r g a n i s a t i o n of k D N A w i t h i n t h e k i n e t o p l a s t in t h e b l o o d f o r m s c o u l d i n v o l v e e i t h e r t h e a s s o c i a t i o n of a n u m b e r of n e t w o r k s o r t h e p r e s e n c e of a l a r g e r s i n g l e n e t w o r k . T h e k D N A netw o r k is p r e s e n t in so l a r g e a m o l e c u l a r c o m p l e x t h a t :to d e t e r m i n e s e d i m e n t a t i o n c o e f f i c i e n t s , the r o t o r s p e e d is l o ' w e r e d to 4000 r p m . I n t h a t c o n d i t i o n the b a n d s e d i m e n t a t i o n of k D N A c a n be m e a s u r e d a c c u r a t e l y (fig. 3). Calcula,tions m a d e of t h e s e d i m e n t a t i o n c o e f f i c i e n t s ( t a b l e I) of k D N A networks from the'blood and culture forms yield e d a v e r a g e v a l u e s of 3730 ± 380 a n d 3'650 _ 200 S r e s p e c t i v e l y ($20 ,w = 5630 a n d 5510 a f t e r c o r r e c t i o n [34]). T h e s e i d e n t i c a l v a l u e s suggest that t h e k D N A n e t w o r k s of ,the b l o o d f o r m s a r e of t h e s a m e size .as t h o s e of t h e c u l t u r e f o r m s . Meas u r e m e n t s w i t h t h e ,electron m i c r o s c o p e of t h e size of k D N A n e t w o r k s f r o m b l o o d stages (fig. 4) s h o w t h a t t h e y are of t h e s a m e size as t h o s e f r o m c u l t u r e f o r m s ('table I), c o n f i r m i n g results o b t a i n e d f r o m s e d i m e n t a t i o n a n a l y s i s . T h e r e is, h o w e v e r , s o m e d i s c r e p a n c y w i t h results o b t a i n e d in b a n d sedimentation analysis with the kDNA networks from intracellular forms. The S values found exper i m e n t a l l y are s i g n i f i c a n t l y h i g h e r t h a n t h o s e obtained with kDNA from blood and culture forms (table I).
Ob3ervations with the electron microscope. T h e s e h a v e s h o w n t h a t the n m l e c u l a r o r g a n i s a tion of t h e k D N A s e e m s to be t h e s a m e in all
BIOCHIMIE, 1978, 60, n ° 4.
l a r g e r c i r c l e s ) . M a n y of t h e m i n i c i r c l e s are l i n k e d to e a c h o t h e r to f o r m l o n g c h a i n s of c a i e n a t e d molecules. T h e n e t w o r k s also c o n t a i n m i n i c i r c l e s join e d to e a c h o t h e r to f o r m figures 'of ,eight o r l a r g e r o l i g o m e r i c m o l e c u l e s . D N A m o l e c u l e s of d i f f e r e n t l e n g t h s , but l a r g e r t h a n m i n i c i r c l e s , are a p p a r e n t at t h e p e r i p h e r y of t h e n e t w o r k s f o r m i n g D N A loops. T h e s e m o l e c u l e s , w h i c h a r e s m a l l e r a n d less n u m e r o u s t h a n t h o s e f o u n d in T. equiperdum El, 211, w e r e o b s e r v e d i n o r e f r e q u e n t l y in b l o o d a n d i n t r a c e l l u l a r f o r m s (about 3 D N A l o o p s p e r n e t w o r k ) t h a n in c u l t u r e f o r m s (tless t h a n 1 D N A l o o p p e r n e t w o r k ) . F i g u r e 4A, s h o w s an e n t i r e net. w o r k f r o m a b l o o d t r y p o m a s t i g o t e ; figure 4B, is a h i g h e r m a g n i f i c a t i o n of t h e z o n e c o n t a i n i n g t h e D N A loops. F i g u r e s 5A a n d B s h o w z o n e s c o n t a i n i n g DNA l o o p s of n e t w o r k s f r o m i n t r a c e l l u l ' a r alnastigotes. In s o m e case, l o o p s a r e s u p e r c o i l e d (fig. 5A). D u r i n g s p r e a d i n g of t h e k D N A n e t w o r k s , s o m e m i n i c i r c l e s are l i b e r a t e d . F r e q u e n c y h i s t o g r a m s of t h e i r c o n t o u r l e n g t h s are g i v e n in figure 6. T h e m e a n c o n t o u r l e n g t h of m i n i c A r c l e s in t h e size r a n g e 0 . 3 9 - 0 . 5 2 ~ m is t h e s a m e in all 3 f o r m s - - 0 . 4 5 ~m, w h i c h c o r r e s p o n d s to 1.450 ± 70 b a s e p a i r s w i t h r e f e r e n c e to t h e s t a n d a r d PM 2 p h a g e DNA. It is a p p a r e n t , e s p e c i ' a l l y in t h e b l o o d and i n t r a c e l l u l a r stages (fig. 6), t h a i t h e r e is d i s c o n t i n u i t y in the d i s t r i b u t i o n of m o n o m e r i c m i n i c i r c l e s (i.e. t h o s e in the Size r a n g e 0 . 3 0 - 0 . 6 5 i.~m), sugg e s t i n g h e t e r o g e n e i t y in m i n i c i r c l e size in kDNA.
G. F. R i o u and W . E. Gutteridge.
370
C i r c u l a r m o l e c u l e s l a r g e r t h a n the m i n i c i r c l e s w e r e o b s e r v e d in t h e b l o o d a n d i n t r a c e l l u l a r f o r m s b u t h a r d l y at all in l h e c u l t u r e f o r m . T h e s e mol, e c u l e s c o u l d be c i r c u l a r d i m e r s a n d o l i g o m e r s w i t h d o u b l e or s e v e r a l t i m e s t h e u s u a l c o n t o u r
l e n g t h of t h e m i n i c i r c l e s . M o r e o v e r a f e w l a r g e r m o l e c u l e s (fig. 5C), h e t e r o g e n e o u s in size, w e r e also o b s e r v e d (7 m o l e c u l e s in b l o o d stage in t h e size r a n g e 2 . 0 - 11.1 ~tm ; 12 m o l e c u l e s i n i n t r a c e l l u l a r stage in t h e size r a n g e 2 . 5 - 1 6 . 3 ~m). T h e s e
Fro. 4. - - Photomicrographs of kDNA from blood stages of T. cruzi. A, entire kDNA network ; B, magnification of the inserted zone with three apparent DNA loops. One minieircle is catenated with the DNA loop at right end.
BIOCH1MIE, 1978, 60, n° 4.
k D N A of T. c r u z i . large molecules are listed in inserts of the histograms (fig. 6). Some m i n i c i r c u l a r kDNA molecules with forks, w h i c h are c o n s i d e r e d to be in replication, w e r e p r e s e n t in the blood and i n t r a c e l l u l a r forms. F i g u r e 5B shows such a m o l e c u l e (arrow) at the p e r i p h e r y of the network.
Agarose gel electrophoresis cleavage of JcDNA.
and
endonuelease
As in ,the T e h u a n t e p e c strain studied p r e v i o u s l y [17, 35], free m i n i c i r c u l a r molecules are p r e s e n t in the kDNA of the Sonya strain. The p r o p o r t i o n of
Fro. 5 . -
free m i n i c i r c l e s was evaluated in the culture forms of both strains after e l e c t r o p h o r e s i s of the [3HI t h y m i d i n e - l a b e l l e d kDNA in 2 p e r cent agarose gels. The results s h o w that about 13 p e r cent of the kDNA of the Sonya s t r a i n is isolated as free m i n i c i r c l e s (column B. table II). El e c t r o p h o r e s i s of labelled kDNA of the blood and inh,acellular forms (Sonya strain) ~vas not u n d e r t a k e n . However, e x p e r i m e n t s p e r f o r m e d w i t h u n l a b e l l e d kDNA s h o w e d the p r e s e n c e of e t e c t r o p h o r e t i c bands of free m i n i c i r c l e s , the p r o p o r t i o n s of w h i c h w e r e s i m i l a r to that of the culture forms. These propor_
Photomicrographs of t~DNA [rom intracellular stages of T. cruzi. DNA was spread by
the formamid technique as described in Materials and Methods. Bar is 0.5 ~m. A, part of a kDNA network with supercoiled loops ; B, part of a kDNA network with a DNA loop and minicircle with a replication fork (arrow) ; C, large circle, 16.3 ~m long, catenated with a dimeric minicircle (arrow).
BIOCHIMIE, 1978, 60, n ° 4.
371
G. F. R i o u a n d W . E. G u t t e r i d g e .
372
kDNA
from
30.
T Cruzi
Culture
(Sonya strain)
form
221 M o l e c u l e s
10
,llll I i!l ,
,
I,
I
Blood form 279
Molecules
in 'pm 2.0 4.1 4,8 7,3 11.1
2 1 1 1 1
in 'pn, 2.5 3.2 5.7 5.8 5.9 6.1 6,6 7,8 12,6 16,3
1 1 I 3 1 1 1 1 1 1
U
-5
3O
.gl
E z
10
,h
f
I
~
L~
Intracellular 284
~,
form
Molecules
30
10
,,.,,
0.½ 5
0.5
h, ,,],ll,I ,
0.7 5
r 1.o
Contour
qength
III
I
I
III I
1.5
in IJm
Fir,. 6. - - Frequency dislribulion histograms of contour lengths of free minicircular molecules of kDNA from culture, blood and intracellular forms of T. cruzi. Molecules listed in i n s e r t s are large circles, h e t e r o g e n e o u s in size, f o u n d in t h e c o r r e s p o n d i n g f o r m s .
BIOCHIM1E, 1978, 60, n ° 4.
o f T. c r u z i .
kDNA
tions were found to be higher w h e n kDNA sampies were stored for several weeks before analysis,
373
kDNA before cleavage with restriction e n d o n u cleases stays in this b a n d (fig. 7 ; lines 7 and 8,
Hpall Eco R 1 Hpall Control Haelll / \ Haelll Ao A
X
B C
Minicircles
D E
F G
cf T
Electrophorelic patterns in a 2 per cent agarose slab gel
(0.1)4 M Tris, 0.02 M Na acetate, 2ram EDTA, pH 7.8; 3V/em for 1~ hr) of restriction endonuclease digests of kDNA from culture forms (ef) of T. cruzi. Columns 1-~e, kDNA from the Tehuantepec strain (T) ; columns 5-7, kDNA, from Sonya strain (S).
cf S
The cleavage p a t t e r n s of the kDNA from the 3 forms of T. c r u z i digested by r e s t r i c t i o n endonucleases a n d revealed by electrophoresis i n 2 per cent agarose gels are p r e s e n t e d in figures 7-9. I n figure 7 the cleavage p a t t e r n s of the culture forms of th,e Sonya strain of T. c r u z i are c o m p a r e d to those of lhe T e h u a n t e p e c strain, p r e v i o u s l y studied i n detail [17, 35, 36]. These cleavage p a t t e r n s are made u p of several e l e c t r o p h o r e t i c bands, designated Ao to G in o r d e r of i n c r e a s i n g mobility. The b a n d s obt,ained with the T e h u a n t e p e c strain were e x a m i n e d by electron m i c r o s c o p y after elul i o n of the DNA from the gel. Band Ao is kDNA w h i c h c a n n o t penetrate into the gel because of the large size of the network. The m a j o r i t y of the BIOCHIMIE, 1978, 60, n ° 4.
Fro. 7 . -
table II). Band A c o n t a i n s l i n e a r molecules 512 !~m long and associations of catenated m i n i circles and b a n d X catenated oligomers (dimers t r i m e r s a n d tetramers) [17]. Band B c o r r e s p o n d s to free m i n i c i r c l e s uncleaved by restriction enzymes. Band C contains l i n e a r DNA molecules of the m i n i c i r c u l a r size. The other bands, D, E, F a n d G, c o n t a i n l i n e a r DNA fragments corresponding to the cleaved m i n i c i r c l e s . The a m o u n t of DNA in each b a n d is presented in table II a n d the r a d i o a c t i v i t y profiles of the electrophoretic patt e r n s are s h o w n in figure 10. Note that the kDNA of the culture forms of both strains gave rise, in most experiments, to tile same n u m b e r of b a n d s h a v i n g the same electrophoretie nlobility, though
G. F. R i o u a n d W . E . G u t t e r i d g e .
374
T h e c l e a v a g e p a t t e r n b y Hpa II o f t h e k D N A i n t h e S o n y a s t r a i n h a d o n e m a i n b a n d (C) a n d m i n o r b a n d s i n t h e D, E a n d F z o n e s (fig. 7, c o l u m n 7
t h e r e w e r e some differences, d e p e n d i n g on r e st r i c t i o n e n z y m e . T h e 4 DNA b a n d s C, D, E a n d F w e r e p r o d u c e d w i t h Hae l I I a n d Eco R1 i n b o t h s t r a i n s .
H p a II
Hae III
Eco R 1 AO A X
D E
F G cf
cf
T
bf
if
S
cf
cf
bf
if cf c f
S
T
T
bf if S
Eleclrophoretic patterns in a 2 per cent agarose slab gel of restriction endonuclease digests of kDNA from the culture forms of the Tehuantepec strain (T) and the culture (cf), blood (bf) and intracellular (if) forms of the Sonya strain (S) of T. cruzi. FIG. 8.
-
-
TABLE II.
Restriction endonuclease cleavage of k D N A f r o m T. c r u z i . Restriction enzyme
Strain ot organism
Radioactivity (per cent) in bands A0
A
X
B
C
D
E
F
G
Haem
T S
1.0 1.0
2.7 1.2
1.0 2.8
2.1 6.3
16.2 39.0
20.8 2.6
39.9 21.6
16.3 24.0
0 1.5
Eco R I
T S
8.0 11.6
5.7 7.7
7.4 6.8
8.1 15.6
20.4 34.2
16.1 7.0
21.1 6.0
12.0 11.1
1.2 0
Hpali
T S
7.1 9.5
4.3 3 8
7.6 7.5
17.1 11.0
62.7 45.3
0 8.2
1.2 8.6
0 5.2
0 0.9
No enzyme
T S
51.0 64.8
11.7 8.8
4.7 4.0
21.8 13.2
10.8 9.2
kDNAs labelled w i t h [ a H ] - t h y m i d i n e f r o m the culture f o r m s of the Sonya (S) and Tehuantepec (T) s t r a i n s were cleaved by restriction endonucleases. Components in the digest were separated electrophoretically (fig. 10) and the radioactivity in each band was determined.
BIOCHIMIE, 1978, 60, n ° 4.
k D N A o f T. c r u z i .
fig. 8, columns 2-4) i n s t e a d of the two ban.ds, (C, E) o b t a i n e d w i t h the T e h u a n t e p e c s t r a i n (fig. 7, c o l u m n 2). The percentage of the various fragment obtained was different in the 2 strains (fig. 10, table II). -
Taq I
Controls
375
and Taql p r o v i d e qualitatively the same cleavage patterns. Similar resul4s have been obtained, however, in other strains of T. cruzi w i t h Hpa II a n d Hae IIl [37]. Double attack of the kDNA successively by Eco R1 and then by Hae l l I yielded
Hindll÷lll Ao
A X Mini_ circles
B C D
E
F cf
bf
if
cf
bf
if
W h e n h y d r o l y s e d by Eco R1 a n d Hae III, the kDNA of the culture, blood a n d i n i r a c e l l u l a r forms of the Sonya strain gives the same n u m b e r of b a n d s w i t h the same electrophoretic m o b i l i t y (fig. 8), though there were quantitative differences (specially 4n the blood form). Quantitative a n d also possibly qualitative ,differences were detected w h e n h y d r o l y s i s was c a r r i e d out w i t h Hpa H (fig. 8), Taq I and H i n d II 4- I,II (fig. 9).
bf
if
Fro. 9. - - Electrophoretic patterns in a 2 per cent agarose slab gel of Taq I and Hind II 4III digests of kDNA from the culture (ef), blood (bf) and intracellular (if) forms of the Sonya strain of T. cruzi. Columns 4-6. kD'NA before cleavage with enzymes (controls),
cf
the same p a t t e r n (fig. l l L i n d i c a t i n g that the enzyme sites for cleavage are located very close to each other. These enzymes were tested at the same time on k and SV 40 DNAs, and p r o v i d e d the usual cleavage fragments.
Discussion.
The sum of the lengths of DNA fragments gener a t e d by the e n d o n u c l e a s e s is greater t h a n the length of the m i n icircle. This f,act cIearly demonstrates that the n u m b e r of e n d o n u c l e a s e sites is not the same for each m i n i c i r c u l a r molecule. It is s u r p r i s i n g , therefore, that Hae Ill, Eco R1, Hpa I1 BIOCHIMIE, 1978, 60, n ° 4.
It is clear from our data that the basic p r o p e r ties of kDNA in all 3 forms of the Sonya strain of T. cruzi are identical, even though the arrangem e n t of that kDNA in the kinetoplast varies a n d
G. F. Riou and W. E. Gutteridge.
376
Hae IH
E c.__£ R1
T
=
2-
m
i
T
~J Eco R1
Hae III 04"
03
0.30.2.
02' 0
01E
o.
!
i
>-
Hpa II
control
T
T
p__~a H II
control
¢=
£ ~E
10-
0,3-
0.2-
03
S
02
5
100
25
0.1-
25
50
75
50
75
100
Slice number
FIG. 10. - - Radioactivitg profiles of restriction enzgme digests of kDNA labelled rvilh L~H]-thymidine from culture forms of the Songa (S) and Tehuan tepee (T) strains of T. cruzi. T h e c o m p o n e n t s i n the d i g e s t s w e r e f r a e t i o n a t e d b y e l e c t r o p h o r e s i s in 2 per cent a g a r o s e gels. B a c k g r o u n d r a d i o a c t i v i t y h a s b e e n deducted.
BIOCHIMIE, 1978, 60, n ° 4.
kDNA
the e n v i r o m n e n t s from w h i c h the parasites were o b t a i n e d were quite distinct. Thus, in each form, the kDNA is in the form of n e t w o r k s w i t h high
o f T. c r u z i .
377
ming, as is likely [see reference 39], that there is the same q u a n t i t y of n u c l e a r DNA in the two stages, lhe kinetoplas¢ of the blood form p r o b a b l y c o n t a i n s two networks. These two n e t w o r k s m u s t be folded since only i n this w a y is it possible to e x p l a i n the four double rows of DNA seen in t h i n sections. Each n e t w o r k is composed of a mass of interl o c k i n g minieir.cular a n d ,apparent l i n e a r molecules. The m e a n c o n t o u r length of the m i n i c i r cular molecules is ,~ 0.45 .~m in all forms a n d m a n y seem attached to each other by c a t e n a t i o n or by f o r m a t i o n of figures-of-eight. These data are s i m i l a r to those o b t a i n e d o r i g i n a l l y w i t h the culture forms of the T e h u a n t e p e c strain. More detailed analysis of some of the properties of the min i c i r c u l a r kDNA of this s t r a i n have been published elsewhere [1, 7]. Some differences w e r e noted at the m o l e c u l a r level between the different forms of the Sonya slain. Dimeric a n d oligomeric circles were present i n the kDNA of the blood a n d i n t r a c e l l u l a r stages in m u c h greater p r o p o r t i o n th'an in the c u p l u r e epimastigote stages ; m i n i c i r c l e s in replication were seen only in ~the blood and i n t r a c e l l u l a r st,ages.
Flt~. 11. - - Eleclrophoretic patterns in 2 per cent agarose slab gel of the products of a doable digestion w i t h Eco R1 and H~aeI I l of kDNA f r o m the culture f o r m s of the S o n y a strain of T. eruzi.
S values w h i c h come r a p i d l y into position i n CsCI gradients a n d have e q u i l i b r i u m densities of 1.6981.699 g / c m 3. The s e d i m e n t a t i o n coefficients for kDNA from c u l t u r e and blood forms are the same a n d are c o m p a r a b l e lo that of the culture form of Tehu~antepec strain, w h i c h is 3 600 + 100 S [38]. The kDNA networks of T. c m l z i have the same size. Since there is p r o p o r t i o n a t e l y more kDNA i n the blood form t h a n in the culture form, assuBIOCH1MIE, 1978, 60, n" 4.
No e v i d e n c e was obtained for the existence of m a x i c i r c l e s in the culture form. In contrast we did isolate a few large circles, heterogeneous in size, from the i n t r a c e l l u l a r and blood stages. In other species of T r y p a n o s o m a t i d a e the few maxici~rcles f o u n d have a constant l e n g t h [10]. However some molecules of i n t e r m e d i a t e c o n t o u r length were described in C r i t h i d i a f a s c i c t t l a t a [40] a n d T. m e g a [41]. The presence of free large circles could be related to the presence of DNA loops observed at the p e r i p h e r y of the networks. If the large circles w e r e a n c h o r e d in the n e t w o r k s they w o u l d only be seen as such after belong released from the n e l w o r k s by m e c h a n i c a l or e n z y m a t i c action. The fact that DNA loops a n d free large circles were f o u n d to be caten ated to m i n i c i r c l e s (fig. 4B a n d 5C) is in favour of this hypothesis. Ho~cever .the large circles heterogeneous i n size we f o u n d in blood a n d i n t r a c e l l u l a r stages of T. e r u z i could have an origin other t h a n from the kinetoplast a n d need not be l i n k e d to the network. The significance of the differences in the gel electrophoretic p a t t e r n s b e t w e e n the various forms of the Sonya s t r a i n is h a r d to ascertain. I n the m a i n these were q u a n t i t a t i v e r a t h e r t h a n qualitative, though there ~Tere differences w i t h i n d i v i dual restriction endonucleases. Differences were also noted b e t w e e n the culture forms of the Sonya 26
G. F . R i o u
3'78
and W. E. Gutteridge.
a n d T e h u a n t e p e c s t r a i n s . S i n c e t h e s e w e r e of t h e s a m e o r d e r as t h a t s e e n b e t w e e n t h e d i f f e r e n t f o r m s of t h e s a m e ( c l o n e d ) s t r a i n , i t i s u n w i s e t o a s c r i b e t h e m to d i f f e r e n c e s i n k D N A b e t w e e n s t r a i n s , as o t h e r a u t h o r s h a v e s u g g e s t e d [37]. T h e h e t e r o g e n e i t y i n n f i n i c i r c l e size r e p o r t e d here for the Sonya strain correlates with other w o r k o n t h e k D N A of t h e c u l t u r e f o r m s of t h e Tehuantepee strain in which heterogeneity in the l e n g t h of t h e m i n i c i r c l e s h a s b e e n o b s e r v e d w h e n t h e k D N A is d i g e s t e d w i t h r e s t r i c t i o n e n z y m e s . F o r e x a m p l e , five size c l a s s e s of m i n i c i r c l e r e s i s t a n t to E c o R1 e x i s t . T h e i r l e n g t h s a r e r e s p e c t i v e l y 0.28 ___ 0.04 .~m, 0.38 _ 0.02 ~m, 0.4.2 ___ 0.02 ¢tm, a n d 0.4.8 ± 0.02 ~ m . H o w e v e r , t h e 0.45 ~ m c l a s s r e p r e s e n t s a b o u t 94 p e r c e n t of t h e m i n i c i r c u l a r population studied in these conditions (unpub l i s h e d d a t a ) . A s i m i l a r size h e t e r o g e n e i t y h a s b e e n f o u n d also i n C r i t h i d i a l u c i l i a e [42]. T h e r e s t r i c t i o n e n z y m e c l e a v a g e of t h e k D N A f r o m t h e t h r e e f o r m s of t h e S o n y a s t r a i n of T. c r u z i y i e l d s s e v e r a l l i n e a r f r a g m e n t s . T h e s u m of t h e i r l e n g t h is n o t t h e s a m e as t h e m o l e c t f l a r w e i g h t of t h e m i nicircles, implying that these molecules are heter o g e n e o u s . B a s e s e q u e n c e h e t e r o g e n e i t y of k D N A m i n , i e i r c l e s is n o w a c c e p t e d as a g e n e r a l c h a r a c t e r i s t i c i n s e v e r a l t r y p a n o s o m e s p e c i e s [17, 42, 43, 44, 45]. It w a s d e m o n s t l ~ a t e d f o r t h e f i r s t t i m e i n T. c r u z i i n t h e T e h u a n t e p e c s t r a i n [36]. It is n o t a r e s u l t of h e t e r o g e n e i t y i n l h e t r y p a n o s o m e p o p u l a t i o n s i n c e s i m i l a r r e s u l t s a r e o b t a i n e d w i t h clon e d a n d u n c l o n e d s t r a i n s [43 a n d t h e s e p r e s e n t investigations].
Aekaowledgements. W e t h a n k Michel Barrois, B r y a n Cover, Mich~le Gabillot and Maria Gaborak for skilled technical assistance. This work was supported in part by a grant to G. B. f r o m the W o r l d Health Organisation, Geneva, S w i t z e r l a n d and in part by grants to W. E. G. f r o m the Ministry of Overseas Development, London, England and the W o r l d Health Organisation, Geneva, S w i t z e r land. Electron micrographs wer~ p e r f o r m e d in the electron microscopy laboratory of Dr E. Delain. REFERENCES. I. Delain, E., Brack, Ch., Lacome, A. & Riou, G. (1972) in Vol. 2. (B r. H. R. L u m s d e n a n d D. A. Evans cd.) in press, Academic Press, New York. 6. Riou, G. & Paoletti, C. (1967) J. Mol. Biol., 28, 377382. 7. Riou, G. & Delain, E. (1969) Proc. Nat. Acad. Sci., 62, 210-217. 8. Riou, G. & Delain, E. (1969) Proc. Nat. Acad. Set., 64, 618-625. 9. Brack, Ch., Delain, E. ~ Riou, G. (1972) Proc. Nat. Acad. Sci., 69, 1642-1646. 10. Steinert, M. & Van Assel, S. (1975) Exptl. Cell Res., 96, 406-~09. 11. Klcisen, C. M., Weislogel, P. O., Fonck, K. ,~ Borst, P. (1976) Eur. J. Biochem., 64, 153-160. 12. Braek, Ch. (1968) in
Comparative study of kinetoplast DNA in cuhure, blood and intracellular forms of Trypanosoma cruzi. (27-2-1978).
* Unitd de Pharmacologie Moldculaire, lnstitut Gustave Roussy, 94800 Villejui[, France. *" Biological Laboratory, University of Kent, Canterbury, Kent, England.
R6sum6.
Summary.
Une 6tude c o m p a r a t i v e du DNA kin6toplastiq u e (kDNA) de ,, f o r m e 1 ,, a 6t6 entreprise sur T r y p a n o s o m a cruz/ & diff~rents s t a d e s de son cycle 6volutif (formes de culture 6pimastigotes, s a n g u i n e s t r y p o m a s t i g o t e s et intracellulaires amastiqotes). Le kDNA de ces t r y p a n o s o m e s p o s s 6 d e des propri6t6s c o m m u n e s (densit6 de flottation 1,698-9 g / c m 3 - - coefficient de s6dim e n t a t i o n $20, w ~ 5500 - - f o r m e m o l 6 c u l a i r e en r 6 s e a u c o m p l e x e de minicercles). Plusieurs diff6rences ont 6t6 not6es. I1 y a proportionnellem e a t p l u s d e kDNA darts les t r y p a n o s o m e s de f o r m e s a n g u i n e ce qui s u q g 6 r e q u e leur kin6top l a s t e r e n f e r m e plusieurs r ~ s e a u x de kDNA p u i s q u e la taille de ces r 6 s e a u x est identique darts les trois formes. Ces r6sultats sont corrobor6s p a r l ' a r r a n g e m e n t ultrastructural du kDNA de la f o r m e s a n g u i n e en 4 d o u b l e couches, diff6rent de celui d e s f o r m e s de culture et intracellulaire en u n e seule d o u b l e couche. Q u e l q u e s g r a n d e s m o l 6 c u l e s circulaires h~t6rog 6 n e s en taille ont 6t6 6 g a l e m e n t o b s e r v 6 e s d a n s les formes s a n q u i n e s et intracellulaires. Apr~s digestion du k D N A p a r les e n d o n u c l e a ses de restriction, les trac6s 61ectrophor6tiques m o n t r e n t q u e l q u e s diff6rences d'ordre quantitatif entre les 3 formes de T. cruz/.
A c o m p a r a l i v e s t u d y h a s b e e n m a d e on the k i n e t o p l a s t DNA (kDNA) form I in culture epimastigote, blood t r y p o m a s t i q o t e a n d intracellular a m a s t i g o t e s t a g e s of T r y p a n o s o m a cruz/. The b a s i c properties of the kDNA in all 3 forms w e r e identical. Thus the DNA w a s in the form of n e t w o r k s of density 1.698-9 g / c m 3 a n d with s e d i m e n t a t i o n coefficients (S20w) of ~ 5500, the n e t w o r k s b e i n g c o m p o s e d of l a r g e c o m p l e x e s of minicircular a n d a p p a r e n t l y linear molecules, the former h a v i n g contour lengths of 0.45 ~m. S e v e r a l differences w e r e noted, The ultrastructural a r r a n g e m e n t of the kDNA in the kinetoplast of the blood s t a g e consisted of three to four double rows of DNA a s c o m p a r e d to one d o u b l e l a y e r e d r o w in the other two stages. There w a s p r o p o r t i o n a t e l y m o r e kDNA in the b l o o d s t a g e s , s u g g e s t i n g that, since the netw o r k s h a v e a p p a r e n t l y the s a m e size (see above), m o r e t h a n one is present, DNA loops situated at the p e r i p h e r y of the kDNA networks w e r e o b s e r v e d in higher proportion in blood a n d intracellular forms. Dimeric a n d oligomeric circles w e r e p r e s e n t in the kDNA of the blood a n d intracellular s t a g e s in m u c h g r e a t e r proportion t h a n in culture e p i m a s t i g o t e s t a g e s . Few l a r g e circular molecules, h e t e r o g e n e o u s in size, w e r e also o b s e r v e d in intracellular b l o o d s t a g e s . There w e r e s o m e differences, m a i n l y quantitative, in the gel electrophoresis p a t t e r n s after e n d o n u c l e a s e digestion.
Introduction.
the base of the flagellum. Electron microscopy has shown that the kinetoplast is a part of the single mitochondrion of these organisms. In the species investigated so far, the kinetoplast has been found to contain DNA which can be distinguished from that in the nucleus by its buoyant density in CsC1 gradients. Kinetoplast DNA (kDNA) can be regar-
Guy F. RIOU ~" and Wins¢on E. GUTTERIDGE +*
A characteristic feature of trypanosomes is the presence of a structure termed the kinetoplast near
To whom all correspondence should be addressed.
366
G. F. R i o u and W . E. Gulteridge.
d e d as the e q u i w a l e n t of m i t o c h o n d r i a l DNA of o t h e r e u k a r y o t i c celIs. R e v i e w s of its p r o p e r t i e s a n d p o s s i b l e f u n c t i o n s h a v e b e e n p u b l i s h e d [1-4]. A c h a r a c t e r i s t i c f e a t u r e of Trltpanosoma cruzi w h i c h d i s t i n g u i s h e s it f r o m A f r i c a n t r y p a n o s o m e s , is t h a t g r o w t h a n d d i v i s i o n in ~he m a m m a l o c c u r i n t r a c e l l u l a r l y , u s u a l l y in t h e m u s c l e t i s s u e of t h e h e a r t a n d , a l i m e n t a r y t r a c t . T h o u g h it is n o w gen e r a l l y a g r e e d t h a t t h e s e i n t r a c e l l u l a r stages h a v e s h o r t f r e e flagella, t h e y a r e r e f e r r e d to as a m a s t i g a t e stages. M o r p h o l o g i c a l l y , t h e y a r e q u i t e distinct from the trypomastigote forms which occur in t h e b l o o d a n d w h i c h , al,though u n a b l e to d i v i d e , s e r v e as a v e h i c u l e b o t h f o r s p r e a d i n g t h e i n f e c t i o n a r o u n d t h e hos~ a n d also f o r i n f e s t i n g the insect vector during feeding. Growth and division in t h e v e c t o r o c c u r m a i n l y as e p i m a s t i g o t e s ; f o r m s s i m i l a r to t h e l a t t e r c a n r e a d i l y be g r o w n in c u l t u r e (see r e f e r e n c e 5 for r e v i e w of t h e biol o g y of t h e o r g a n i s m ) . E a r l i e r w o r k in V i l l e j u i f sho"wed t h a t the cult u r e e p i m a s t i g o t e f o r m s of T. cruzi c o n t a i n a v e r y h i g h p r o p o r t i o n of k D N A (20-25 p e r c e n t of t h e total cell DNA) [6]. It w a s also s h o w n t h a t t h i s k D N A w a s a p p a r e n t l y in t h e f o r m of an a s s o c i a t i o n of l i n e a r m o l e c u l e s a n d m i n i c i r c u l a r ,molecules ( c o n t o u r l e n g t h 0.45 i~m) w h i c h t h u s f o r m e d a c o m p l e x n e t w o r k [1, 7], T h e r e a r e 20-30,000 of t h e m i n i c i r c u l a r m o l e c u l e s p e r k i n e t o p l a s t [81. T h e i r r e p l i c a t i o n ,appeared to be as d e s c r i b e d b y C a i r n s f o r the c h r o m o s o m e s of Escherichia colt [9], It is not k n o w n w h a t g e n e t i c i n f o r m a t i o n t h e y contain. A s i m i l a r o r g a n i s a t i o n of k D N A w a s l a t e r f o u n d in oth, er s p e c i e s of t r y p ~ n o s o m e s ( r e v i e w e d i n ref. 2), t h o u g h m o s t r e c e n t s t u d i e s [10, 11, r e v i e w e d i n 4] suggest t h a t at least in s o m e s p e c i e s of Crithidia a n d Trgpanosoma, t h e l i n e a r k D N A m o l e c u l e s i~n f a c t o c c u r as m a x i c i r c l e s ( f r o m 6 to 11 ~ m c o n t o u r l e n g t h d e p e n d i n g on s p e c i e s ) . It has b e e n s u g g e s t e d t h a t t h e s e r e p r e s e n t t h e t r u e m i t o c h o n d r i a l D N A of t h e s e o r g a n i s m s . T h e r e is g o o d e v i d e n c e t h a t t h e b l o o d t r y p o m a s t i g o t e stages of T. cruzi, in c o n t r a s t to t h e b l o o d stages of A f r i c a n t r y p a n o s o m e s , c a t a b o l i s e g l u c o s e in l a r g e p a r t to GO2 via a T,CA c y c l e a n d respiratory chain ; the mitochondri,a are cristate, TCA c y c l e s e n z y m e s a r e p r e s e n t a n d e y t o c h r o m e s h a v e b e e n d e s c r i b e d [ r e v i e w e d in 5]. I t is l i k e l y t h e r e f o r e t h a t w h a t e v e r g e n e t i c i n f o r m a t i o n is c o n t a i n e d w i t h i n t h e k ] ) N A it is :expressed in t h e s e stages. F u r t h e r m o r e , p r e l i m i : n a r y u l t r a s t r u c t u r a l s t u d i e s in ¥ i l l e j u i f a.nd in o t h e r l a b o r a t o r i e s [12, 13] of b l o o d t r y p o m a s t i g o t e f o r m s s h o w e d t h a t t h e
BIOCH1M1E, 1978, 60, n ° 4.
o r g a n i s a t i o n of t h e DN,A in t h e kinetopl,ast w a s q u i t e d i s t i n c t f r o m t h a t in t h e c u l t u r e f o r m s . At this t i m e , m e t h o d s h a d b e e n d e v e l o p e d in C a n t e r b u r y to i s o l a t e b l o o d t r y p o m a s t i g o t e a n d i n t r a e e l l u l a r a m a s t i g o t e stages f r o m i n f e c t e d r a i s [14]. It w a s d e c i d e d , t h e r e f o r e , to c a r r y out a j o i n t i n v e s t i g a t i o n of t h e k D N A of t h e c u l t u r e e p i m a s t i g o t e , blood trypomastigote and intracellular amastigote f o r m s of T. cruzi. A p r e l i m i n a r y a c c o u n t of s o m e of t h e s e r e s u l t s has b e e n p r e s e n t e d [15].
Materials
and
Methods.
Organism. The Sonya strain of Trgpanosoma cruzi was used, except where there is a clear indication to the contrary. Most of the experiments involving restriction endonucleases were carried out after it had been cloned (Cover and Gutteridge, in preparation). The strain was maintained in Porton-derived XL mice by syringe passage (subcutaneous inoculation) at intervals of 2-3 weeks. Parasites for experiments were obtained from rats (wt range 100-120 g) which had been subjected ,~ 14, days earlier to whole body ~-irradiation (580 rads) prior to inoculation sub-cutaneously with 107 blood trypomastigotes from mice [14]. Culture epimastigote forms were grown at 28°C in a modified LIT m e d i u m [16]. In a few experiments, clearly indicated, culture forms of the Tehuantepec strain [17] were also used. When labelled DNA was required, 5 ~ C i / m l of [methyl-3H]-thymidine (specific activity 20: Ci/mM) was added to medium at the beginning of the culture period. Isolation of blood stages. The method used [14] was based on the DEAE cellulose column method described by Lanham and Godfrey [18]. It involved the use of their phosphate saline glucose buffer at a pH value of 7.5 and at an ionie strength of 0.2~)6 and was carried out at room temperature. The m a j o r i t y of the trypanosomes were eluted withou t retardation as a m a j o r peak. No contamination with red cells, white cells or platelets could he observed by light microscopy. As judged by light microscopy, organisms were motile and normal in form ; they were. infective to mice. Isolation of intracellular stages. Isolation was made from rat skeletal muscle by a p r o c e d u r e described elsewhere [14]. This involves disruption of the muscle tissue in an MSE tissue homogeniser, filtration through gauze and then incubation of the homogenate with deoxyribonuclease, collagenase and trypsin to destroy muscle cell nuclei, break down gelatin-like proteins and degrade muscle cell debris to small particles. A pellet fraction is th~en obtained by differential centrifugation contaminated only by a few white (1-2 per cent) and red (2-11) per cent) cells. In most preparations, these blood cells were removed by centrifugation in a linear sucrose gradient (0.25.-0.70 M). Between 1 and 2 per cent of the parasites thus isolated are epimastigotes and trypomastigotes ; the remainder are amastigotes. Parasites isolated by this method are apparently undamaged as judged by light and electron microscopy and are metabolically active at least as far as respiration and nucleic acid and protein syn-
kDNA
o[ T. cruzi.
t h e s i s are concerned. The i n f e c t i v i t y for mice of b o t h purified p r e p a r a t i o n s a n d i n i t i a l cell h o m o g e n a t e s ean be a c c o u n t e d for by t h e e p i m a s t i g o t e s a n d t r y p o m a s t i gores p r e s e n t in t h e m .
Electron microscopy. P r e p a r a t i o n s were fixed w i t h g l u t a r a l d e h y ~ e - o s m i u m t e t r a o x i d e a n d e m b e d d e d in Epon. T h i n sections w e r e d o u b l e - s t a i n e d w i t h u r a n y l acetate a n d lead citrate as described p r e v i o u s l y [19].
Extraction of total cell DNA. T h i s w a s e x t r a c t e d by a m o d i f i c a t i o n of t h e m e t h o d of M a r m u r [20, 21]. T r y p a n o z o m e s , a f t e r l y s i s in s a r k o s y l (final c o n c e n t r a t i o n 1 per cent w / v ) i n SSC (NaC1, 0.15 M ; Nas citrate, 0.015 M, pH 7.4) were digested w i t h p r o n a s e (13 grade, Calbioehem, 1 m g / m l ) for 2 h r at 37~C (the stock solut i o n of t h e e n z y m e (10 m g / m l ) w a s self-digested for 2 h r at 37°C in SSC). The digest w a s t h e n e x t r a c t e d once w i t h c h l o r o f o r m / i s o a m y l alcohol (24:1 by vol). The a q u e o u s phase was dialysed extensively against SSC a n d t h e n i n c u b a t e d a t 37°C, first w i t h RNase A (pre-treat.ed by h e a t i n g to 90°C for 11) rain) at a conc e n t r a t i o n o f 100 ,~g/ml for ~ hr, t h e n w i t h RNase TI (10 U / m l ) a n d finally w i t h p r o n a s e , T h e DNA w a s e x t r a c t e d a g a i n w i t h organic s o l v e n t s as above a n d red i a l y s e d a g a i n s t 0.1 S~SC, Fraciionalion of kDNA. Total DNA e x t r a c t s were f r a e t i o n a t e d b y u l t r a e e n t r i f u g a t i o n in a p r o p i d i u m diiodide-(PErI)-CsC1 g r a d i e n t (PI)I 51~)igxg/ml ; 50 Ti R o t o r ; 24 h r ; 20'°C; 40,(~00 r p m ) [22]. T h e lower b a n d w h i c h c o n t a i n s kDNA in its c o n s t r a i n e d f o r m was collected a n d t h e PDI w a s e x t r a c t e d w i t h i s o p r o p a n o l [23]. The kl)NA w a s freed of r e s i d u a l n u c l e a r DN~A by s u b s e q u e n t u l t r a e e n t r i f u g a t i o n in a H'g÷+ --Cs2SO, g r a d i e n t [6], The kDNA f o r m I w a s u s e d in e x p e r i m e n t s described in t h i s s t u d y ,
367
w a s l a y e r e d on distilled w a t e r a s a h y p o p h a s e a t 4°C. DNA f r o m P I ~ p h a g e (9,90~ n u c l e o t i d e s pairs, 6.4 X 1~6 d a l t o n s ) w a s u s e d as i n t e r n a l size s t a n d a r d .
A n a l y t i c a l ultraeentri[uyation. B u o y a n t d e n s i t y e x p e r i m e n t s were p e r f o r m e d at 25°C for 24 h r at 44,770 r p m i n a B e c k m a n m o d e l E c e n t r i f u g e . The b u o y a n t d e n s i t y g r a d i e n t of V i n o g r a d a n d H e a r s t [24] w a s u s e d in t h e e a I e u l a t i o n of t h e r e s u l t s w i t h reference to E. colt DNA (density 0 = 1.710 g/eroS). T h e DNA m a r k e r w a s 2C phage DNA of Bacillus subtilis (0 = 1.742 g/em3). T h e s e d i m e n t a t i o n coefficients of kDNA were m e a s u r e d by b a n d s e d i m e n t a t i o n a n a l y s i s i n 3 M CsC1 0.01 M Nas-EDTA (pH 8.0) according to t h e m e t h o d of V i n o g r a d et al [25] except t h a t a lower speed of r o t a t i o n w a s u s e d (4000 r p m at 20°C in a B e c k m a n ANJ rotor). Each cell w a s filled w i t h 15 lal of the DNA s o l u t i o n p r e v i o u s l y d i a l y s e d a g a i n s t 2 M NaCl-O.O,1 M Na~-EDrfA. R e s t r i c t i o n endonucleases cleavaye of kDNA. Several r e s t r i c t i o n e n z y m e s were used to e x a m i n e t h e overall c o m p o s i t i o n of kDNA. Eco RI e n d o n u c l e a s e w a s p u r i fied e s s e n t i a l l y according to Y o s h i m o r i [26]. H i n d II A-Ill a n d Hae IH e n d o n u c l e a s e s were respectively purified b y t h e m e t h o d s of S m i t h a n d Wilcox [27] a n d Middleton et al. [28] Hpa II e n d o n u e l e a s e , p r e p a red as described b y S h a r p e t a l [29], w a s k i n d l y provided b y P. N a r d e u x . T a q I p r e p a r e d b y t h e m e t h o d described by Sato et al. [30] w a s a g e n e r o u s gift of Dr. J. F e u n t e u n . T h e activities of l h e v a r i o u s re'strietion e n z y m e s were checked u n d e r c o n d i t i o n s described in the c o r r e s p o n d i n g references, u s i n g ~ DNA a n d SV 40 DNA. T h e DNA f r a g m e n t s of kDNA were s e p a r a t e d by e l e c t r o p h o r e s i s in vertical 2 per cent a g a r o s e slab gels as described by D a n n a et al. [31]. After m i g r a t i o n ,
F16. 1. - - Photomicrographs of thin sections of the S o n y a strain of T. cruzi s h o w i n y detail of the kinctoplast. A, eultfire e p i m a s t i g o t e ; B, blood t r y p o m a s t i g o t e ; C, i n t r a e e l l u l a r anaastigote.
Preparation ol kDNA for electron microscopy, kI>NA m o l e c u l e s w e r e e x a m i n e d u n d e r a P h i l i p s EM 300 elect r o n microscope a f t e r being s p r e a d w i t h o u t f o r m a m i d [1] or w i t h f o r m a m i d as described p r e v i o u s l y [7] h u t w i t h t h e following modifications. 55 ~I of DNA solut i o n in SSC were m i x e d w i t h ¢5 lxl of f o r m a m i d (Fluka) a n d 15 ]xl of c y t o c h r o m e c (1 m g / m l ) (Calbioehem). T h e m i x t u r e was m a i n t a i n e d at 4°C. T h e DNA BIOCHIMIE, 1978, 60, n ° 4,
t h e y were located by e t h i d i u m b r o m i d e s t a i n i n g a n d p h o t o g r a p h e d u n d e r UV l i g h t w i t h a red filter. W h e n labelled DNA w a s used, t h e r a d i o a c t i v i t y of each b a n d w a s d e t e r m i n e d a f t e r slicing t h e gel into 1 r a m t h i c k slices. The slices were dissolved o v e r n i g h t in 1 ml of Soluene 350 (Packard) at 37°C. S c i n t i l l a t i o n f l u i d w a s added a n d slices were e s t i m a t e d for r a d i o a c t i v i t y in a liquid s c i n t i l l a t i o n s p e c t r o m e t e r .
G. F. R i o u a n d W . E. G u t t e r i d g e .
368
Ultracentrifugation in CsCl.
Results. K inetoplast ultrastructure. E x a m i n a t i o n of thin sections of the 3 forms of T. cruzi c o n f i r m e d results d e s c r i b e d p r e v i o u s l y about the u l t r a s t r u c t u r a l c o n f o r m a t i o n of kDNA
Culture
U l t r a c e n t r i f u g a t i o n in i s o p y c n i c CsC1 gradients s h o w e d the p r e s e n c e in all 3 f o r m s of 2 DNA components : a n u c l e a r c o m p o n e n t of density 1.710 g / c m 3 ( ~ 51 per c e n t G + C) and a rapidb a n d i n g k i n e t o p l a s t c o m p o n e n t of density 1.6981.699 g / c m 3 (-~ 40 per cent G + C) (fig. 2). The
Intracellular
Blood f o r m
form
form
1.698
1.710
1
1.742
l
1.710
1.742 1.742 1.698 1.698 1.710 FI6. 2. - - Isopycnic ultracentrifugation analysis of DNA from T. eruzi. Phage 2 G DNA from Bacillus subtilis was used as a marker (5 ---- 1.7'42 g/cm3. 9 ---- 1.710 g/era3 was nuclear DNA. ~ = 1.69,8 g/cm3 was kDNA).
in isolated culture epimastigote [19] and b l o o d t r y p o m a s t i g o t e [123 forms and the s t r u c t u r e of the kinetoplast in i n t r a c e l l u l a r ,amastigote stages e x a m i n e d in situ [13, 32, 33]. In the culture forms, the kinetoplast is localized at the base of the flagellum adjac.ent to the basal body. It is b o u n d to m i t 0 c h o n d r i a l m e m b r a n e s . The kDNA appears as a double r o w of regular and w e l l d e f i n e d elect r o n dense m a t e r i a l (fig. 1A). In the blood forms the kinetoplast is globular and the kDNA is arranged in 3 or 4 double l a y e r e d r o w s of fibers (fig. 1B). In the i n t r a c e l l u l a r forms several k i n d s of figures are obserwed for the k i n e t o p l a s t and its DNA. The most frequent, N 70 p e r cent of thin sections observed, show a s t r u c t u r e of kDNA s i m i l a r to that of the e p i m a s t i g o t e forms, t h o u g h w i t h a less o r d e r e d fibrillar a r r a n g e m e n t (fig. 1C). The kDNA found in other figures is a r r a n g e d in several double r o w s and appears to c o r r e s p o n d to an i n t e r m e d i a t e state b e t w e e n the t r y p o m a s t i g o t e and the epimastigote forms.
BIOCH1MIE, 1978, 60, n ° 4.
results of calculations f r o m the u l t r a c e n t r i f u g a t i o n data about the p r o p o r t i o n s of kDNA in the differ e n t forms of T. cruzi are given in table I. As
FIG. 3. - - Band sedimentation analysis of kDNA from the blood forms of T. eruzi in 3 M CsCl at !tOO0 rpm, Photographs were taken at 2 rain intervals.
k D N A of T . c r u z i . e x p e c t e d f r o m e l e c t r o n m i c r o g r a p h s of u l t r a t h i n s e c t i o n s , t h e r e is p r o p o r t i o n a t e l y m o r e k D N A in t h e b l o o d stages t h a n in t h e t~ther 2 stages.
369
3 stages. It is in t h e f o r m of a c o m p l e x n e t w o r k of m a n y t h o u s a n d s of m i n i c i r c u l a r m o l e c u l e s and a p p a r e n t l o n g l i n e a r p i e c e s ( w h i c h m a y b e p a r t of
TABLE I.
Properties of kDNA in the different forms of T. cruzi. Form of T. cruzi.
Culture Blood Intraeellular
kDNA as per cent of total DNA
20.3 ~ 0.60 3 3 . 6 -~- 4.1 2 6 . 0 ~__ 2 . 0
(4) (7) (4)
Sedimentation coefficients of kDNA networks (in S)
Size ol kDNA networks (l~m'-') •
3650 -t- 200 (3) 3730 ~- 380 (3) 4510 and 4550
121 ~__ 18 (13) 125 ~__ 26 (17) 128 -4- 23 (17)
Values for the percentage of kDNA were derived from densitometer tracings obtained after analytical ultraeentrifugation in CsC1 gradients of total DNA as described in figure 2. To calculate their size the kDNA networks were spread without formamid as described in [1]. Electron mierographs were all taken with the same magnification in order to abtain the entire network in the centre of the plate. Numbers in brackets are numbers of experiments done or networks measured. The sedimentation coefficients and the sizes of the kDNA networks were determined from k D N A form I as described in Materials and Methods.
T h e o r g a n i s a t i o n of k D N A w i t h i n t h e k i n e t o p l a s t in t h e b l o o d f o r m s c o u l d i n v o l v e e i t h e r t h e a s s o c i a t i o n of a n u m b e r of n e t w o r k s o r t h e p r e s e n c e of a l a r g e r s i n g l e n e t w o r k . T h e k D N A netw o r k is p r e s e n t in so l a r g e a m o l e c u l a r c o m p l e x t h a t :to d e t e r m i n e s e d i m e n t a t i o n c o e f f i c i e n t s , the r o t o r s p e e d is l o ' w e r e d to 4000 r p m . I n t h a t c o n d i t i o n the b a n d s e d i m e n t a t i o n of k D N A c a n be m e a s u r e d a c c u r a t e l y (fig. 3). Calcula,tions m a d e of t h e s e d i m e n t a t i o n c o e f f i c i e n t s ( t a b l e I) of k D N A networks from the'blood and culture forms yield e d a v e r a g e v a l u e s of 3730 ± 380 a n d 3'650 _ 200 S r e s p e c t i v e l y ($20 ,w = 5630 a n d 5510 a f t e r c o r r e c t i o n [34]). T h e s e i d e n t i c a l v a l u e s suggest that t h e k D N A n e t w o r k s of ,the b l o o d f o r m s a r e of t h e s a m e size .as t h o s e of t h e c u l t u r e f o r m s . Meas u r e m e n t s w i t h t h e ,electron m i c r o s c o p e of t h e size of k D N A n e t w o r k s f r o m b l o o d stages (fig. 4) s h o w t h a t t h e y are of t h e s a m e size as t h o s e f r o m c u l t u r e f o r m s ('table I), c o n f i r m i n g results o b t a i n e d f r o m s e d i m e n t a t i o n a n a l y s i s . T h e r e is, h o w e v e r , s o m e d i s c r e p a n c y w i t h results o b t a i n e d in b a n d sedimentation analysis with the kDNA networks from intracellular forms. The S values found exper i m e n t a l l y are s i g n i f i c a n t l y h i g h e r t h a n t h o s e obtained with kDNA from blood and culture forms (table I).
Ob3ervations with the electron microscope. T h e s e h a v e s h o w n t h a t the n m l e c u l a r o r g a n i s a tion of t h e k D N A s e e m s to be t h e s a m e in all
BIOCHIMIE, 1978, 60, n ° 4.
l a r g e r c i r c l e s ) . M a n y of t h e m i n i c i r c l e s are l i n k e d to e a c h o t h e r to f o r m l o n g c h a i n s of c a i e n a t e d molecules. T h e n e t w o r k s also c o n t a i n m i n i c i r c l e s join e d to e a c h o t h e r to f o r m figures 'of ,eight o r l a r g e r o l i g o m e r i c m o l e c u l e s . D N A m o l e c u l e s of d i f f e r e n t l e n g t h s , but l a r g e r t h a n m i n i c i r c l e s , are a p p a r e n t at t h e p e r i p h e r y of t h e n e t w o r k s f o r m i n g D N A loops. T h e s e m o l e c u l e s , w h i c h a r e s m a l l e r a n d less n u m e r o u s t h a n t h o s e f o u n d in T. equiperdum El, 211, w e r e o b s e r v e d i n o r e f r e q u e n t l y in b l o o d a n d i n t r a c e l l u l a r f o r m s (about 3 D N A l o o p s p e r n e t w o r k ) t h a n in c u l t u r e f o r m s (tless t h a n 1 D N A l o o p p e r n e t w o r k ) . F i g u r e 4A, s h o w s an e n t i r e net. w o r k f r o m a b l o o d t r y p o m a s t i g o t e ; figure 4B, is a h i g h e r m a g n i f i c a t i o n of t h e z o n e c o n t a i n i n g t h e D N A loops. F i g u r e s 5A a n d B s h o w z o n e s c o n t a i n i n g DNA l o o p s of n e t w o r k s f r o m i n t r a c e l l u l ' a r alnastigotes. In s o m e case, l o o p s a r e s u p e r c o i l e d (fig. 5A). D u r i n g s p r e a d i n g of t h e k D N A n e t w o r k s , s o m e m i n i c i r c l e s are l i b e r a t e d . F r e q u e n c y h i s t o g r a m s of t h e i r c o n t o u r l e n g t h s are g i v e n in figure 6. T h e m e a n c o n t o u r l e n g t h of m i n i c A r c l e s in t h e size r a n g e 0 . 3 9 - 0 . 5 2 ~ m is t h e s a m e in all 3 f o r m s - - 0 . 4 5 ~m, w h i c h c o r r e s p o n d s to 1.450 ± 70 b a s e p a i r s w i t h r e f e r e n c e to t h e s t a n d a r d PM 2 p h a g e DNA. It is a p p a r e n t , e s p e c i ' a l l y in t h e b l o o d and i n t r a c e l l u l a r stages (fig. 6), t h a i t h e r e is d i s c o n t i n u i t y in the d i s t r i b u t i o n of m o n o m e r i c m i n i c i r c l e s (i.e. t h o s e in the Size r a n g e 0 . 3 0 - 0 . 6 5 i.~m), sugg e s t i n g h e t e r o g e n e i t y in m i n i c i r c l e size in kDNA.
G. F. R i o u and W . E. Gutteridge.
370
C i r c u l a r m o l e c u l e s l a r g e r t h a n the m i n i c i r c l e s w e r e o b s e r v e d in t h e b l o o d a n d i n t r a c e l l u l a r f o r m s b u t h a r d l y at all in l h e c u l t u r e f o r m . T h e s e mol, e c u l e s c o u l d be c i r c u l a r d i m e r s a n d o l i g o m e r s w i t h d o u b l e or s e v e r a l t i m e s t h e u s u a l c o n t o u r
l e n g t h of t h e m i n i c i r c l e s . M o r e o v e r a f e w l a r g e r m o l e c u l e s (fig. 5C), h e t e r o g e n e o u s in size, w e r e also o b s e r v e d (7 m o l e c u l e s in b l o o d stage in t h e size r a n g e 2 . 0 - 11.1 ~tm ; 12 m o l e c u l e s i n i n t r a c e l l u l a r stage in t h e size r a n g e 2 . 5 - 1 6 . 3 ~m). T h e s e
Fro. 4. - - Photomicrographs of kDNA from blood stages of T. cruzi. A, entire kDNA network ; B, magnification of the inserted zone with three apparent DNA loops. One minieircle is catenated with the DNA loop at right end.
BIOCH1MIE, 1978, 60, n° 4.
k D N A of T. c r u z i . large molecules are listed in inserts of the histograms (fig. 6). Some m i n i c i r c u l a r kDNA molecules with forks, w h i c h are c o n s i d e r e d to be in replication, w e r e p r e s e n t in the blood and i n t r a c e l l u l a r forms. F i g u r e 5B shows such a m o l e c u l e (arrow) at the p e r i p h e r y of the network.
Agarose gel electrophoresis cleavage of JcDNA.
and
endonuelease
As in ,the T e h u a n t e p e c strain studied p r e v i o u s l y [17, 35], free m i n i c i r c u l a r molecules are p r e s e n t in the kDNA of the Sonya strain. The p r o p o r t i o n of
Fro. 5 . -
free m i n i c i r c l e s was evaluated in the culture forms of both strains after e l e c t r o p h o r e s i s of the [3HI t h y m i d i n e - l a b e l l e d kDNA in 2 p e r cent agarose gels. The results s h o w that about 13 p e r cent of the kDNA of the Sonya s t r a i n is isolated as free m i n i c i r c l e s (column B. table II). El e c t r o p h o r e s i s of labelled kDNA of the blood and inh,acellular forms (Sonya strain) ~vas not u n d e r t a k e n . However, e x p e r i m e n t s p e r f o r m e d w i t h u n l a b e l l e d kDNA s h o w e d the p r e s e n c e of e t e c t r o p h o r e t i c bands of free m i n i c i r c l e s , the p r o p o r t i o n s of w h i c h w e r e s i m i l a r to that of the culture forms. These propor_
Photomicrographs of t~DNA [rom intracellular stages of T. cruzi. DNA was spread by
the formamid technique as described in Materials and Methods. Bar is 0.5 ~m. A, part of a kDNA network with supercoiled loops ; B, part of a kDNA network with a DNA loop and minicircle with a replication fork (arrow) ; C, large circle, 16.3 ~m long, catenated with a dimeric minicircle (arrow).
BIOCHIMIE, 1978, 60, n ° 4.
371
G. F. R i o u a n d W . E. G u t t e r i d g e .
372
kDNA
from
30.
T Cruzi
Culture
(Sonya strain)
form
221 M o l e c u l e s
10
,llll I i!l ,
,
I,
I
Blood form 279
Molecules
in 'pm 2.0 4.1 4,8 7,3 11.1
2 1 1 1 1
in 'pn, 2.5 3.2 5.7 5.8 5.9 6.1 6,6 7,8 12,6 16,3
1 1 I 3 1 1 1 1 1 1
U
-5
3O
.gl
E z
10
,h
f
I
~
L~
Intracellular 284
~,
form
Molecules
30
10
,,.,,
0.½ 5
0.5
h, ,,],ll,I ,
0.7 5
r 1.o
Contour
qength
III
I
I
III I
1.5
in IJm
Fir,. 6. - - Frequency dislribulion histograms of contour lengths of free minicircular molecules of kDNA from culture, blood and intracellular forms of T. cruzi. Molecules listed in i n s e r t s are large circles, h e t e r o g e n e o u s in size, f o u n d in t h e c o r r e s p o n d i n g f o r m s .
BIOCHIM1E, 1978, 60, n ° 4.
o f T. c r u z i .
kDNA
tions were found to be higher w h e n kDNA sampies were stored for several weeks before analysis,
373
kDNA before cleavage with restriction e n d o n u cleases stays in this b a n d (fig. 7 ; lines 7 and 8,
Hpall Eco R 1 Hpall Control Haelll / \ Haelll Ao A
X
B C
Minicircles
D E
F G
cf T
Electrophorelic patterns in a 2 per cent agarose slab gel
(0.1)4 M Tris, 0.02 M Na acetate, 2ram EDTA, pH 7.8; 3V/em for 1~ hr) of restriction endonuclease digests of kDNA from culture forms (ef) of T. cruzi. Columns 1-~e, kDNA from the Tehuantepec strain (T) ; columns 5-7, kDNA, from Sonya strain (S).
cf S
The cleavage p a t t e r n s of the kDNA from the 3 forms of T. c r u z i digested by r e s t r i c t i o n endonucleases a n d revealed by electrophoresis i n 2 per cent agarose gels are p r e s e n t e d in figures 7-9. I n figure 7 the cleavage p a t t e r n s of the culture forms of th,e Sonya strain of T. c r u z i are c o m p a r e d to those of lhe T e h u a n t e p e c strain, p r e v i o u s l y studied i n detail [17, 35, 36]. These cleavage p a t t e r n s are made u p of several e l e c t r o p h o r e t i c bands, designated Ao to G in o r d e r of i n c r e a s i n g mobility. The b a n d s obt,ained with the T e h u a n t e p e c strain were e x a m i n e d by electron m i c r o s c o p y after elul i o n of the DNA from the gel. Band Ao is kDNA w h i c h c a n n o t penetrate into the gel because of the large size of the network. The m a j o r i t y of the BIOCHIMIE, 1978, 60, n ° 4.
Fro. 7 . -
table II). Band A c o n t a i n s l i n e a r molecules 512 !~m long and associations of catenated m i n i circles and b a n d X catenated oligomers (dimers t r i m e r s a n d tetramers) [17]. Band B c o r r e s p o n d s to free m i n i c i r c l e s uncleaved by restriction enzymes. Band C contains l i n e a r DNA molecules of the m i n i c i r c u l a r size. The other bands, D, E, F a n d G, c o n t a i n l i n e a r DNA fragments corresponding to the cleaved m i n i c i r c l e s . The a m o u n t of DNA in each b a n d is presented in table II a n d the r a d i o a c t i v i t y profiles of the electrophoretic patt e r n s are s h o w n in figure 10. Note that the kDNA of the culture forms of both strains gave rise, in most experiments, to tile same n u m b e r of b a n d s h a v i n g the same electrophoretie nlobility, though
G. F. R i o u a n d W . E . G u t t e r i d g e .
374
T h e c l e a v a g e p a t t e r n b y Hpa II o f t h e k D N A i n t h e S o n y a s t r a i n h a d o n e m a i n b a n d (C) a n d m i n o r b a n d s i n t h e D, E a n d F z o n e s (fig. 7, c o l u m n 7
t h e r e w e r e some differences, d e p e n d i n g on r e st r i c t i o n e n z y m e . T h e 4 DNA b a n d s C, D, E a n d F w e r e p r o d u c e d w i t h Hae l I I a n d Eco R1 i n b o t h s t r a i n s .
H p a II
Hae III
Eco R 1 AO A X
D E
F G cf
cf
T
bf
if
S
cf
cf
bf
if cf c f
S
T
T
bf if S
Eleclrophoretic patterns in a 2 per cent agarose slab gel of restriction endonuclease digests of kDNA from the culture forms of the Tehuantepec strain (T) and the culture (cf), blood (bf) and intracellular (if) forms of the Sonya strain (S) of T. cruzi. FIG. 8.
-
-
TABLE II.
Restriction endonuclease cleavage of k D N A f r o m T. c r u z i . Restriction enzyme
Strain ot organism
Radioactivity (per cent) in bands A0
A
X
B
C
D
E
F
G
Haem
T S
1.0 1.0
2.7 1.2
1.0 2.8
2.1 6.3
16.2 39.0
20.8 2.6
39.9 21.6
16.3 24.0
0 1.5
Eco R I
T S
8.0 11.6
5.7 7.7
7.4 6.8
8.1 15.6
20.4 34.2
16.1 7.0
21.1 6.0
12.0 11.1
1.2 0
Hpali
T S
7.1 9.5
4.3 3 8
7.6 7.5
17.1 11.0
62.7 45.3
0 8.2
1.2 8.6
0 5.2
0 0.9
No enzyme
T S
51.0 64.8
11.7 8.8
4.7 4.0
21.8 13.2
10.8 9.2
kDNAs labelled w i t h [ a H ] - t h y m i d i n e f r o m the culture f o r m s of the Sonya (S) and Tehuantepec (T) s t r a i n s were cleaved by restriction endonucleases. Components in the digest were separated electrophoretically (fig. 10) and the radioactivity in each band was determined.
BIOCHIMIE, 1978, 60, n ° 4.
k D N A o f T. c r u z i .
fig. 8, columns 2-4) i n s t e a d of the two ban.ds, (C, E) o b t a i n e d w i t h the T e h u a n t e p e c s t r a i n (fig. 7, c o l u m n 2). The percentage of the various fragment obtained was different in the 2 strains (fig. 10, table II). -
Taq I
Controls
375
and Taql p r o v i d e qualitatively the same cleavage patterns. Similar resul4s have been obtained, however, in other strains of T. cruzi w i t h Hpa II a n d Hae IIl [37]. Double attack of the kDNA successively by Eco R1 and then by Hae l l I yielded
Hindll÷lll Ao
A X Mini_ circles
B C D
E
F cf
bf
if
cf
bf
if
W h e n h y d r o l y s e d by Eco R1 a n d Hae III, the kDNA of the culture, blood a n d i n i r a c e l l u l a r forms of the Sonya strain gives the same n u m b e r of b a n d s w i t h the same electrophoretic m o b i l i t y (fig. 8), though there were quantitative differences (specially 4n the blood form). Quantitative a n d also possibly qualitative ,differences were detected w h e n h y d r o l y s i s was c a r r i e d out w i t h Hpa H (fig. 8), Taq I and H i n d II 4- I,II (fig. 9).
bf
if
Fro. 9. - - Electrophoretic patterns in a 2 per cent agarose slab gel of Taq I and Hind II 4III digests of kDNA from the culture (ef), blood (bf) and intracellular (if) forms of the Sonya strain of T. cruzi. Columns 4-6. kD'NA before cleavage with enzymes (controls),
cf
the same p a t t e r n (fig. l l L i n d i c a t i n g that the enzyme sites for cleavage are located very close to each other. These enzymes were tested at the same time on k and SV 40 DNAs, and p r o v i d e d the usual cleavage fragments.
Discussion.
The sum of the lengths of DNA fragments gener a t e d by the e n d o n u c l e a s e s is greater t h a n the length of the m i n icircle. This f,act cIearly demonstrates that the n u m b e r of e n d o n u c l e a s e sites is not the same for each m i n i c i r c u l a r molecule. It is s u r p r i s i n g , therefore, that Hae Ill, Eco R1, Hpa I1 BIOCHIMIE, 1978, 60, n ° 4.
It is clear from our data that the basic p r o p e r ties of kDNA in all 3 forms of the Sonya strain of T. cruzi are identical, even though the arrangem e n t of that kDNA in the kinetoplast varies a n d
G. F. Riou and W. E. Gutteridge.
376
Hae IH
E c.__£ R1
T
=
2-
m
i
T
~J Eco R1
Hae III 04"
03
0.30.2.
02' 0
01E
o.
!
i
>-
Hpa II
control
T
T
p__~a H II
control
¢=
£ ~E
10-
0,3-
0.2-
03
S
02
5
100
25
0.1-
25
50
75
50
75
100
Slice number
FIG. 10. - - Radioactivitg profiles of restriction enzgme digests of kDNA labelled rvilh L~H]-thymidine from culture forms of the Songa (S) and Tehuan tepee (T) strains of T. cruzi. T h e c o m p o n e n t s i n the d i g e s t s w e r e f r a e t i o n a t e d b y e l e c t r o p h o r e s i s in 2 per cent a g a r o s e gels. B a c k g r o u n d r a d i o a c t i v i t y h a s b e e n deducted.
BIOCHIMIE, 1978, 60, n ° 4.
kDNA
the e n v i r o m n e n t s from w h i c h the parasites were o b t a i n e d were quite distinct. Thus, in each form, the kDNA is in the form of n e t w o r k s w i t h high
o f T. c r u z i .
377
ming, as is likely [see reference 39], that there is the same q u a n t i t y of n u c l e a r DNA in the two stages, lhe kinetoplas¢ of the blood form p r o b a b l y c o n t a i n s two networks. These two n e t w o r k s m u s t be folded since only i n this w a y is it possible to e x p l a i n the four double rows of DNA seen in t h i n sections. Each n e t w o r k is composed of a mass of interl o c k i n g minieir.cular a n d ,apparent l i n e a r molecules. The m e a n c o n t o u r length of the m i n i c i r cular molecules is ,~ 0.45 .~m in all forms a n d m a n y seem attached to each other by c a t e n a t i o n or by f o r m a t i o n of figures-of-eight. These data are s i m i l a r to those o b t a i n e d o r i g i n a l l y w i t h the culture forms of the T e h u a n t e p e c strain. More detailed analysis of some of the properties of the min i c i r c u l a r kDNA of this s t r a i n have been published elsewhere [1, 7]. Some differences w e r e noted at the m o l e c u l a r level between the different forms of the Sonya slain. Dimeric a n d oligomeric circles were present i n the kDNA of the blood a n d i n t r a c e l l u l a r stages in m u c h greater p r o p o r t i o n th'an in the c u p l u r e epimastigote stages ; m i n i c i r c l e s in replication were seen only in ~the blood and i n t r a c e l l u l a r st,ages.
Flt~. 11. - - Eleclrophoretic patterns in 2 per cent agarose slab gel of the products of a doable digestion w i t h Eco R1 and H~aeI I l of kDNA f r o m the culture f o r m s of the S o n y a strain of T. eruzi.
S values w h i c h come r a p i d l y into position i n CsCI gradients a n d have e q u i l i b r i u m densities of 1.6981.699 g / c m 3. The s e d i m e n t a t i o n coefficients for kDNA from c u l t u r e and blood forms are the same a n d are c o m p a r a b l e lo that of the culture form of Tehu~antepec strain, w h i c h is 3 600 + 100 S [38]. The kDNA networks of T. c m l z i have the same size. Since there is p r o p o r t i o n a t e l y more kDNA i n the blood form t h a n in the culture form, assuBIOCH1MIE, 1978, 60, n" 4.
No e v i d e n c e was obtained for the existence of m a x i c i r c l e s in the culture form. In contrast we did isolate a few large circles, heterogeneous in size, from the i n t r a c e l l u l a r and blood stages. In other species of T r y p a n o s o m a t i d a e the few maxici~rcles f o u n d have a constant l e n g t h [10]. However some molecules of i n t e r m e d i a t e c o n t o u r length were described in C r i t h i d i a f a s c i c t t l a t a [40] a n d T. m e g a [41]. The presence of free large circles could be related to the presence of DNA loops observed at the p e r i p h e r y of the networks. If the large circles w e r e a n c h o r e d in the n e t w o r k s they w o u l d only be seen as such after belong released from the n e l w o r k s by m e c h a n i c a l or e n z y m a t i c action. The fact that DNA loops a n d free large circles were f o u n d to be caten ated to m i n i c i r c l e s (fig. 4B a n d 5C) is in favour of this hypothesis. Ho~cever .the large circles heterogeneous i n size we f o u n d in blood a n d i n t r a c e l l u l a r stages of T. e r u z i could have an origin other t h a n from the kinetoplast a n d need not be l i n k e d to the network. The significance of the differences in the gel electrophoretic p a t t e r n s b e t w e e n the various forms of the Sonya s t r a i n is h a r d to ascertain. I n the m a i n these were q u a n t i t a t i v e r a t h e r t h a n qualitative, though there ~Tere differences w i t h i n d i v i dual restriction endonucleases. Differences were also noted b e t w e e n the culture forms of the Sonya 26
G. F . R i o u
3'78
and W. E. Gutteridge.
a n d T e h u a n t e p e c s t r a i n s . S i n c e t h e s e w e r e of t h e s a m e o r d e r as t h a t s e e n b e t w e e n t h e d i f f e r e n t f o r m s of t h e s a m e ( c l o n e d ) s t r a i n , i t i s u n w i s e t o a s c r i b e t h e m to d i f f e r e n c e s i n k D N A b e t w e e n s t r a i n s , as o t h e r a u t h o r s h a v e s u g g e s t e d [37]. T h e h e t e r o g e n e i t y i n n f i n i c i r c l e size r e p o r t e d here for the Sonya strain correlates with other w o r k o n t h e k D N A of t h e c u l t u r e f o r m s of t h e Tehuantepee strain in which heterogeneity in the l e n g t h of t h e m i n i c i r c l e s h a s b e e n o b s e r v e d w h e n t h e k D N A is d i g e s t e d w i t h r e s t r i c t i o n e n z y m e s . F o r e x a m p l e , five size c l a s s e s of m i n i c i r c l e r e s i s t a n t to E c o R1 e x i s t . T h e i r l e n g t h s a r e r e s p e c t i v e l y 0.28 ___ 0.04 .~m, 0.38 _ 0.02 ~m, 0.4.2 ___ 0.02 ¢tm, a n d 0.4.8 ± 0.02 ~ m . H o w e v e r , t h e 0.45 ~ m c l a s s r e p r e s e n t s a b o u t 94 p e r c e n t of t h e m i n i c i r c u l a r population studied in these conditions (unpub l i s h e d d a t a ) . A s i m i l a r size h e t e r o g e n e i t y h a s b e e n f o u n d also i n C r i t h i d i a l u c i l i a e [42]. T h e r e s t r i c t i o n e n z y m e c l e a v a g e of t h e k D N A f r o m t h e t h r e e f o r m s of t h e S o n y a s t r a i n of T. c r u z i y i e l d s s e v e r a l l i n e a r f r a g m e n t s . T h e s u m of t h e i r l e n g t h is n o t t h e s a m e as t h e m o l e c t f l a r w e i g h t of t h e m i nicircles, implying that these molecules are heter o g e n e o u s . B a s e s e q u e n c e h e t e r o g e n e i t y of k D N A m i n , i e i r c l e s is n o w a c c e p t e d as a g e n e r a l c h a r a c t e r i s t i c i n s e v e r a l t r y p a n o s o m e s p e c i e s [17, 42, 43, 44, 45]. It w a s d e m o n s t l ~ a t e d f o r t h e f i r s t t i m e i n T. c r u z i i n t h e T e h u a n t e p e c s t r a i n [36]. It is n o t a r e s u l t of h e t e r o g e n e i t y i n l h e t r y p a n o s o m e p o p u l a t i o n s i n c e s i m i l a r r e s u l t s a r e o b t a i n e d w i t h clon e d a n d u n c l o n e d s t r a i n s [43 a n d t h e s e p r e s e n t investigations].
Aekaowledgements. W e t h a n k Michel Barrois, B r y a n Cover, Mich~le Gabillot and Maria Gaborak for skilled technical assistance. This work was supported in part by a grant to G. B. f r o m the W o r l d Health Organisation, Geneva, S w i t z e r l a n d and in part by grants to W. E. G. f r o m the Ministry of Overseas Development, London, England and the W o r l d Health Organisation, Geneva, S w i t z e r land. Electron micrographs wer~ p e r f o r m e d in the electron microscopy laboratory of Dr E. Delain. REFERENCES. I. Delain, E., Brack, Ch., Lacome, A. & Riou, G. (1972) in Vol. 2. (B r. H. R. L u m s d e n a n d D. A. Evans cd.) in press, Academic Press, New York. 6. Riou, G. & Paoletti, C. (1967) J. Mol. Biol., 28, 377382. 7. Riou, G. & Delain, E. (1969) Proc. Nat. Acad. Sci., 62, 210-217. 8. Riou, G. & Delain, E. (1969) Proc. Nat. Acad. Set., 64, 618-625. 9. Brack, Ch., Delain, E. ~ Riou, G. (1972) Proc. Nat. Acad. Sci., 69, 1642-1646. 10. Steinert, M. & Van Assel, S. (1975) Exptl. Cell Res., 96, 406-~09. 11. Klcisen, C. M., Weislogel, P. O., Fonck, K. ,~ Borst, P. (1976) Eur. J. Biochem., 64, 153-160. 12. Braek, Ch. (1968) in
