15.10. 1975
Speeialia
1149
electrons) w h e n u n c o a t e d p r e p a r a t i o n s are e x a m i n e d . I n a d d i t i o n t h e m e t a l l i c m a r k e r p e r m i t s good s p a t i a l resolut i o n t o b e a c h i e v e d in S E M ~0.
a t t h e surface of Candida utilis cells was localized b y stabilized colloidal gold g r a n u l e s c o a t e d w i t h e i t h e r a n t i m a n n a n a n t i b o d i e s or Con A.
Summary. A r a p i d m e t h o d h a s b e e n d e v e l o p e d t o visualize cell surface r e c e p t o r s in t h e SEM. T h u s m a n n a n
M. HORISBERGER, JAQUELINEROSSET a n d H. BAUER
10 Acknowledgment. We thank Mrs M. WEBER for the photographic work.
Research Department, NestH Products Technical Assistance Co. Ltd., CH-1814 La Tour-de-Peilz (Switzerland), 16 June 1975.
Morphology of Colloidal Gold, Ferritin and Anti-Ferritin Antibody Complexes E l e c t r o n m i c r o s c o p i c a l s t u d i e s on t h e f o r m a t i o n of ferritin - anti-ferritin complexes have been published p r e v i o u s l y 1-a. Colloidal gold - ferritin m i x t u r e s h a v e also b e e n used to d e m o n s t r a t e m e m b r a n e holes in o s m o t i c a n d s a p o n i n h e m o l y s e s a n d m e m b r a n e lesions in i m m u n e lysis 4, 5. H o w e v e r , t h e s t r u c t u r e of t h e c o m p l e x e s f o r m e d b e t w e e n colloidal gold particles, t e r r i t i n a n d a n t i - f e r r i t i n a n t i b o d i e s h a s n o t b e e n s t u d i e d in detail. I t was t h e r e f o r e i n t e r e s t i n g to i n v e s t i g a t e t h e m o r p h o l o g y of t h e s e complexes in o r d e r to e v a l u a t e t h e i r p o t e n t i a l use as m a r k e r s for t r a n s m i s s i o n - a n d s c a n n i n g - e l e c t r o n microscope c y t o c h e m i c a l studies. Experimental. T h e anti-ferritii1 a n t i b o d i e s were isolated b y i m m u n o a d s o r p t i o n f r o m a colostral l a c t o s e r u m of a cow i m m u n i z e d w i t h h o r s e spleen f e r r i t i n ( c a d m i u m free, 2 • c r y s t a l l i z e d ; P e n t e x Biochemicals). T h e p r e s e n c e of p r e c i p i t a t i n g a n t i - f e r r i t i n a n t i b o d i e s was s h o w n b y a p o s i t i v e ring t e s t 6 a g a i n s t a f e r r i t i n s o l u t i o n (1 mg/ml). F e r r i t i n a n d a n t i - f e r r i t i n a n t i b o d i e s were a d s o r b e d o n t o colloidal gold a c c o r d i n g t o t h e p r o c e d u r e of FAULK a n d TAYLOR 7, m o d i f i e d b y GERBER et al. s. T h e colloids were s u s p e n d e d in p h o s p h a t e - b u f f e r e d saline, p H 7.2 t o a final a b s o r b a n c e of 3.6 a t 520 n m . T h e d i f f e r e n t p r e p a r a t i o n s were e x a m i n e d in a Philips E M 300 e l e c t r o n microscope a f t e r n e g a t i v e s t a i n i n g w i t h 5% aqueous uranyl-acetate. Results. I n Figures 1-3 t h e i n d i v i d u a l m a t e r i a l s used in o u r e x p e r i m e n t s are r e p r e s e n t e d . T h e n o n - s t a b i l i z e d colloidal gold p a r t i c l e s t e n d to a g g r e g a t e (Figure 1). T h e y h a v e a p o l y h e d r i c a l f o r m (inset) a n d are u n s t a b l e under the electron beam. With progressive beam radiation, t h e y b e c o m e e l e c t r o n t r a n s p a r e n t (mass loss). T h e h o r s e s p l e e n ferritin (Figure 2) is q u i t e h o m o g e n e o u s ; h o w e v e r , some d e t a c h e d a p o f e r r i t i n ( p r o t e i n shell of t h e f e r r i t i n molecule) c a n be o b s e r v e d o c c a s i o n a l l y (arrow). T h e p u r i f i e d a n t i - f e r r i t i n a n t i b o d i e s (Figure 3) f o r m s m a l l clusters of fairly c o n s t a n t d i a m e t e r (30-40 nm). Aggregates of v a r i a b l e sizes of f e r r i t i n m a r k e d w i t h colloidal gold g r a n u l e s (average d i a m e t e r 5.2 n m ) are v i s u a l i z e d in F i g u r e 4. O v e r 9 5 % of t h e f e r r i t i n molecules are labelled w i t h a t least one gold particle. A d s o r p t i o n of t h e gold colloid t a k e s place o n t o t h e p r o t e i n p a r t of t h e f e r r i t i n molecules. F r e e a p o f e r r i t i n is t h e r e f o r e labelled as well. Figure 5 illustrates the purified anti-ferritin antibodies m a r k e d w i t h colloidal gold. M o s t i m p o r t a n t is t h e o b s e r v a t i o n t h a t all colloid p a r t i c l e s are c o v e r e d b y a n t i b o d i e s , i.e. a f t e r n e g a t i v e s t a i n i n g a clear, e l e c t r o n - t r a n s p a r e n t zone is visible a r o u n d all colloidal gold granules. I n f a v o u r a b l e p r o j e c t i o n s , t h e Y - s h a p e of t h e a n t i - f e r r i t i n a n t i b o d i e s a d s o r b e d o n t o t h e colloid p a r t i c l e s c a n b e r e v e a l e d (Figure 5 simple a r r o w a n d inset). I t is e v i d e n t t h a t t h i s a r r a n g e m e n t w i t h t h e colloid g r a n u l e s in t h e center, s u r r o u n d e d b y a d s o r b e d i m m u n p r o t e i n , is m o s t f a v o u r a b l e for i m m u n o c y t o c h e m i c a l r e a c t i o n s . S m a l l
clusters of free a n t i b o d i e s ( c o m p a r e w i t h F i g u r e 3) are recognized o c c a s i o n a l l y (double arrows). C o m p l e x e s b e t w e e n f e r r i t i n a n d colloidal gold, c o a t e d w i t h a n t i f e r r i t i n a n t i b o d i e s , are seen in F i g u r e 6. T h e y a p p e a r , d u e to t h e p r e s e n c e of a n t i b o d i e s , less d e n s e l y p a c k e d t h a n t h o s e f o r m e d b y f e r r i t i n a n d u n c o a t e d colloidal gold. I n t h i s p r e p a r a t i o n , r a t h e r i m p o r t a n t f l u c t u a t i o n s in t h e size of t h e colloid g r a n u l e s occurred. F i n a l l y , t h e r a t h e r heterogeneous complexes formed between ferritin marked w i t h colloidal gold a n d colloidal gold c o v e r e d w i t h a n t i f e r r i t i n a n t i b o d i e s are d e m o n s t r a t e d in F i g u r e s 7 a n d 8. W i t h a d e q u a t e c o n c e n t r a t i o n s of f e r r i t i n a n d a n t i f e r r i t i n a n t i b o d i e s , c o m p l e x e s of t h e t y p e seen in F i g u r e 7 are o b t a i n e d . All f e r r i t i n molecules are s u r r o u n d e d b y colloid particles, b u t i t is i m p o s s i b l e to d i s t i n g u i s h bet w e e n t h e gold g r a n u l e s d i r e c t l y a d s o r b e d to t h e t e r r i t i n and those linked to the ferritin via the anti-ferritin antibodies. I n F i g u r e 7, t h e f e r r i t i n a n d a n t i - f e r r i t i n a n t i b o d i e s are n o t r e v e a l e d b e c a u s e n o n e g a t i v e s t a i n i n g was applied. N e v e r t h e l e s s , t h e f e r r i t i n n u c l e u s (simple arrow) is easily d i s t i n g u i s h e d f r o m t h e gold p a r t i c l e s (double arrow) b y its c o n s i d e r a b l y lower d e n s i t y . T h e s a m e c o m p l e x e s a f t e r n e g a t i v e s t a i n i n g w i t h u r a n y l a c e t a t e are s h o w n in F i g u r e 8. A g a i n all t h e f e r r i t i n molecules are s u r r o u n d e d b y colloid granules. T h e t y p i c a l f e r r i t i n s t r u c t u r e is m a s k e d (arrow) d u e t o a d s o r p t i o n of a n t i b o d i e s . T h i s is in agreem e n t w i t h o b s e r v a t i o n s o n f e r r i t i n - a n t i - f e r r i t i n complexes 2. Discussion. F r o m t h e e l e c t r o n m i c r o g r a p h s p r e s e n t e d i t is e v i d e n t t h a t m i x e d c o m p l e x e s are v e r y h e t e r o g e n e o u s in size a n d t h e r e f o r e n o t s u i t a b l e as c y t o c h e m i c a l m a r k e r s . Two o b s e r v a t i o n s are, h o w e v e r , of i m p o r t a n c e . F i r s t l y p r o t e i n molecules are a d s o r b e d a r o u n d colloidal gold g r a n u l e s in a s t e r i c a l l y f a v o u r a b l e p o s i t i o n n e c e s s a r y for c y t o c h e m i c a l c o u p l i n g or for t h e a n t i b o d y - a n t i g e n react i o n . T h i s p r o p e r t y h a s a l r e a d y b e e n successfully e x p l o i t e d in labelling e x p e r i m e n t s in t h e t r a n s m i s s i o n -7-1~ a n d
1 A. FEINSTEIN and A. J. ROWE, Nature, Lond. 205, 147 (1965). 2 j. p. ROBINSON, J. molee. Biol. 17, 456 (1966). a j. p. ROBINSONand S. S. SCHUFFMAN,Immunology 20, 883 (1971). 4 p. SEEMAN, D. CHENG and G. H. ILES, J. Cell Biol. 56, 519 (1973). G. H. ILES, P. SEEMAN, P. NAVLOR and B. C[NADER, J. Cell Biol. 56, 528 (1973). 6 D. H. CAMPBELL,J. S. GARVEY,N. E. CREMER and D. H. SUSSDORF, in Methods in Immunology (W. A. Benjamin Inc., New York 1964), p. 131. W. P. FAULKand G. M. TAYLOR, Immunochemistry 8, 1081 (1971). 8 H. GERBER, M. HORISBERCmRand H. BAUER, Infect. hnmunity 7, 487 (1973). H. BAUER, M. HORISBERGER,D. A. BUSH and E. SIGARLAKIE, Arch. Mikrobiol. 85, 202 (1972). 10 H. BAUER,D. R. FARR and M. HORISBERGER,Arch. Microbiol. 97, 17 (1974).
All electron micrographs, except Figure 7 (unstained), are from material negatively stained with 5% UO~-acetate. The dimensions on the micrographs are given in ran. Figs. 1-3. Individual materials used in our experiments. 1. Non-stabilized gold colloid particles showing instability under the electron beam. The polyhedrical form of the individual granules is well revealed (arrow, inset), x 250,000; inset x 500,000. 2. The horse spleen ferritin preparation is quite homogeneous. Some free apoferritin is indicated by an arrow. • 250,000. 3. The anti-ferritin antibodies tend to form small clusters of fairly constant diameter ( 3 0 4 0 nm). • 250,000. Figs. 4 + 5. Direct marking of ferritin and anti-ferritin antibodies with colloidal gold. 4. The complexes formed between ferritin directly marked with colloidal gold are rather heterogeneous. Over 95% of the ferritin nmleeules are marked with at least one gold particle. • 250,000. 5. All colloid particles are coated with anti-ferritin antibodies. In favourable projections (arrow, inset), the Y-shape of the antibodies is revealed. The double arrow points to small cluster of free antibodies (eomp. Figure 3). x 250,000; inset • 500,000. Figs. 6-8. Marking of native and gold-labelled ferritin with anti-ferritin antibodies coated colloidal gold. 6. Complex formed between ferritin and colloidal gold coated with anti-ferritin antibodies. • 250,000. 7. Complexes formed between gold-labelled ferritin and colloidal gold coated with anti-ferritin antibodies. No negative staining was applied and therefore the protein part of the ferritin molecules and the anti-ferritin antibodies around the gold colloids is not revealed. Ferritin nucleus: 4; gold particles: ~ . x 250,000. 8. Complexes formed between gold-labelled ferritin and colloidal gold coated with anti-ferritin antibodies. This preparation contains an excess of free antibodies, and it appears that free antibodies attach preferentially to the ferritin molecules (arrow). • 250,000.
15.10. 1975
Specialia
r e c e n t l y in t h e s c a n n i n g - u e l e c t r o n microscope. Secondly, e v e n w i t h o u t n e g a t i v e staining, t h e gold colloid a n d t h e f e r r i t i n molecules (iron nucleus) can easily be d e t e c t e d a n d d i s t i n g u i s h e d in t h e t r a n s m i s s i o n e l e c t r o n microscope. D o u b l e labelling e x p e r i m e n t s , using colloidal gold p a r t i c l e s coated with a protein (phytohemagglutinin, antibodies,
etc.) a n d f e r r i t i n - p r o t e i n c o n j u g a t e s can t h e r e f o r e be performed simultaneously.
Summary. T h e m o r p h o l o g y of m o d e l c o m p l e x e s bet w e e n colloidal gold, ferritin a n d a n t i - f e r r i t i n a n t i b o d i e s h a s b e e n s t u d i e d in o r d e r t o e v a l u a t e t h e p o t e n t i a l of colloidal gold as a c y t o c h e m i c a l m a r k e r . I-I. BAUER, H. GERBER 12 a n d
11 M. HORISBERGER,J. Ross~r and H. BAUER,submitted for publication. 12 Pathology Institute, University of Bern, CH-3008 Bern, Switzerland. la Acknowledgment. We thank Prof. H. ISLIKER, Biochemistry Institute, University of Lausanne, for the bovine colostral antiserum against ferritin.
H y b r i d i z a t i o n of M i t o c h o n d r i a l a n d C y t o p l a s m i c Mitochondrial and Nuclear DNA
1151
M. HORISBERGER13
Research Department, Nestld Products Technical Assistance Co. Ltd., CH-1814 La Tour-de-Peilz (Switzerland), and Pathology Institute, University of Berne, CH-3008 Berne (Switzerland), 16 June 1975.
Ribosomal
T h e genetic origins of m i t o c h o n d r i a l a n d c y t o p l a s m i c r R N A s f r o m r a t liver were i n v e s t i g a t e d b y R N A - D N A h y b r i d i z a t i o n e x p e r i m e n t s . I t was s h o w n in earlier s t u d i e s ~ t h a t m i t o c h o n d r i a l r R N A s f r o m r a t or m o u s e liver were d i f f e r e n t f r o m t h e i r c y t o p l a s m i c c o u n t e r p a r t s in several of t h e i r p h y s i c o - c h e m i c a l p r o p e r t i e s . If in a d d i t i o n m i t o c h o n d r i a l r R N A s h y b r i d i z e specifically w i t h m i t o c h o n d r i a l D N A a n d n o t w i t h n u c l e a r DNA, t h i s w o u l d be an indication of a d i f f e r e n t genetic origin f r o m b o t h t y p e s of r R N A s . 2V[aterial and methods. M i t o c h o n d r i a a n d R N A were p r e p a r e d f r o m a d u l t r a t liver as d e s c r i b e d in earlier s t u d i e s 1. D u r i n g subcellular f r a c t i o n a t i o n a n d R N A e x t r a c t i o n , 2 ribonuclease i n h i b i t o r s were used to aw)id R N A d e g r a d a t i o n : a n a t u r a l one p r e p a r e d f r o m r a t liver the d a y before, a n d d i e t h y l p y r o c a r b o n a t e . A f t e r e t h a n o l p r e c i p i t a t i o n , t h e R N A s were purified b y 2 c e n t r i f u g a t i o n s t h r o u g h 5 20% sucrose g r a d i e n t s in order to completely eliminate tRNAs. T h e c y t o p l a s m i c r i b o s o m a l R N A s were p r e p a r e d a c c o r d i n g to STEVENIN e t al. 2, e x c e p t t h a t for t h e ribonuclease t r e a t m e n t , 0.05 ~*g ribonuclease per ml for 20 m i n a t 0 ~ was used to r e m o v e m R N A . M i t o c h o n d r i a l D N A was p r e p a r e d according to lx'rEL et al. a a n d t h e n u c l e a r I ) N A according to MARMUR 4, e x c e p t t h a t D N A was p r e c i p i t a t e d b y cold e t h a n o l a n d n o t b y isopropanol. B o t h D N A s were f r a g m e n t e d b y s o n i c a t i o n as d e s c r i b e d b y MORI et al. 5, in o r d e r to o b t a i n f r a g m e n t s w i t h a l e n g t h of a b o u t 500 base pairs, w h i c h c o r r e s p o n d s a p p r o x i m a t i v e l y to a gene size6, L The t w o
RNA with
species of D N A s were t r i t i a t e d in v i t r o w i t h s o d i u m a H - b o r o h y d r i d e as d e s c r i b e d b y LEE a n d GORDONS. T h e specific activities of D N A s o b t a i n e d v a r i e d f r o m 5-1500 cpm/tzg DNA. The m e l t i n g t e m p e r a t u r e s (Tms) of t h e D N A f r a g m e n t s a n d h y b r i d s were d e t e r m i n e d b y t h e r m i c elutions a t increasing t e m p e r a t u r e s on h y d r o x y a p a t i t e columns. This m e t h o d of t e m p e r a t u r e m e a s u r e m e n t s 5 is v e r y r a p i d and several d e t e r m i n a t i o n s can be d o n e s i m u l t a n e o u s l y . H y d r o x y a p a t i t e was p r e p a r e d a c c o r d i n g t o TISELIUS 9 a n d m o d i f i e d b y LEVIN ~~ T h e elution was p e r f o r m e d w i t h 0.12 M s o d i u m p h o s p h a t e buffer. Nuclease $1 was p r e p a r e d a c c o r d i n g t o SUTTON 11 in our l a b o r a t o r y f r o m t a k a d i a s t a s e . E n z y m a t i c a c t i v i t y was d e t e r m i n e d a c c o r d i n g to SUTTON n e x c e p t t h a t t h e i n c u b a t i o n was p e r f o r m e d a t 37 ~ a n d n o t a t 56 ~ This enzyme preferentially attacks single-stranded DNA. H y b r i d i z a t i o n c o n d i t i o n s were t h o s e of our l a b o r a t o r y . T r i t i a t e d D N A (12,000-15,000 cpm) was i n c u b a t e d w i t h a large excess of R N A (100 to 600 times) in o r d e r to decrease s t r o n g l y I ) N A - D N A reassociations, in 0.75 M s o d i u m p h o s p h a t e b u f f e r for 48 h a t 65~ a f t e r h e a t d e n a t u r a t i o n . H y d r o x y a p a t i t e (2 ml per column) was 1 A. I)IERICU, M. }". ITTEL and M. WINTZERITtI, l.ife Sci. 13, 553 (1973). 2 J. STEVENIN, J. SAMEC, M. JACOl~ and P. MANDEL, J. molec.
Biol. 33, 777 (1968).
a M. E. ITT~:r., M. WINTZERITH and P. MANnEL, C. r. Acad. Sci., Paris, Sdrie D 274, 3622 (1972). 4 J. MARMUR,J. molee. Biol. 3, 208 (1961}. 5 K. MORI, M. WINTZERITH and P. ~[ANDEL, Biochilnie 54, 1427
"Fable I. Hybridization percentages of mitochondrial and cytoplasmic rRNAs with mitochondrial and nuclear DNA
MitochondrialrRNA Cytoplasmic rRNA
Mitoehondrial DNA (% DNAhybridized)
Nuclear DNA (% DNA hybridized)
1.74 (10) 0.02 (3)
0.01 0.42
(3) (8)
The values in brackets are the number of detertninations.
(1972). 6 R. J. BRITTEN and D. E. KOtlNE, Science 161, 529 (1968). 7 E. H. DAVIDSONand B. R. HOUGH, Proe. natn. Aead. Sci., USA 63, 342 (1969}. s V. F. LEE and M. P. GORDON, Biochim. biophys. Aeta 2,38, 174 (1971). 9 A. TISELIUS, S. HJERTISNand O. LEVlN, Arch. Biochem. Biophys. 65, 132 (1956). 10 O. LEVIN, Methods in Enzymology (Eds. S. P. COLOWICKand N. O. KAPLAN; Acadeufie Press, New York 1962), vol. 5, p. 27. 11 W. D. SUTTON, Bioehim. biophys. Aeta 2,f0, 522 (1971).
Table ii. Temperature values frolu nuclear and mitochoudrial DNA, and RNA-DNA hybrids Temperature (~ Mitochondrial DNA Mitoehondrial DNA-mitochondrial rRNA hybrids
82 74.8
Temperature (~ Nuclear DNA Nuclear DNA-cytoplaslnic~rRNA hybrids
86 78.5
Speeialia
1149
electrons) w h e n u n c o a t e d p r e p a r a t i o n s are e x a m i n e d . I n a d d i t i o n t h e m e t a l l i c m a r k e r p e r m i t s good s p a t i a l resolut i o n t o b e a c h i e v e d in S E M ~0.
a t t h e surface of Candida utilis cells was localized b y stabilized colloidal gold g r a n u l e s c o a t e d w i t h e i t h e r a n t i m a n n a n a n t i b o d i e s or Con A.
Summary. A r a p i d m e t h o d h a s b e e n d e v e l o p e d t o visualize cell surface r e c e p t o r s in t h e SEM. T h u s m a n n a n
M. HORISBERGER, JAQUELINEROSSET a n d H. BAUER
10 Acknowledgment. We thank Mrs M. WEBER for the photographic work.
Research Department, NestH Products Technical Assistance Co. Ltd., CH-1814 La Tour-de-Peilz (Switzerland), 16 June 1975.
Morphology of Colloidal Gold, Ferritin and Anti-Ferritin Antibody Complexes E l e c t r o n m i c r o s c o p i c a l s t u d i e s on t h e f o r m a t i o n of ferritin - anti-ferritin complexes have been published p r e v i o u s l y 1-a. Colloidal gold - ferritin m i x t u r e s h a v e also b e e n used to d e m o n s t r a t e m e m b r a n e holes in o s m o t i c a n d s a p o n i n h e m o l y s e s a n d m e m b r a n e lesions in i m m u n e lysis 4, 5. H o w e v e r , t h e s t r u c t u r e of t h e c o m p l e x e s f o r m e d b e t w e e n colloidal gold particles, t e r r i t i n a n d a n t i - f e r r i t i n a n t i b o d i e s h a s n o t b e e n s t u d i e d in detail. I t was t h e r e f o r e i n t e r e s t i n g to i n v e s t i g a t e t h e m o r p h o l o g y of t h e s e complexes in o r d e r to e v a l u a t e t h e i r p o t e n t i a l use as m a r k e r s for t r a n s m i s s i o n - a n d s c a n n i n g - e l e c t r o n microscope c y t o c h e m i c a l studies. Experimental. T h e anti-ferritii1 a n t i b o d i e s were isolated b y i m m u n o a d s o r p t i o n f r o m a colostral l a c t o s e r u m of a cow i m m u n i z e d w i t h h o r s e spleen f e r r i t i n ( c a d m i u m free, 2 • c r y s t a l l i z e d ; P e n t e x Biochemicals). T h e p r e s e n c e of p r e c i p i t a t i n g a n t i - f e r r i t i n a n t i b o d i e s was s h o w n b y a p o s i t i v e ring t e s t 6 a g a i n s t a f e r r i t i n s o l u t i o n (1 mg/ml). F e r r i t i n a n d a n t i - f e r r i t i n a n t i b o d i e s were a d s o r b e d o n t o colloidal gold a c c o r d i n g t o t h e p r o c e d u r e of FAULK a n d TAYLOR 7, m o d i f i e d b y GERBER et al. s. T h e colloids were s u s p e n d e d in p h o s p h a t e - b u f f e r e d saline, p H 7.2 t o a final a b s o r b a n c e of 3.6 a t 520 n m . T h e d i f f e r e n t p r e p a r a t i o n s were e x a m i n e d in a Philips E M 300 e l e c t r o n microscope a f t e r n e g a t i v e s t a i n i n g w i t h 5% aqueous uranyl-acetate. Results. I n Figures 1-3 t h e i n d i v i d u a l m a t e r i a l s used in o u r e x p e r i m e n t s are r e p r e s e n t e d . T h e n o n - s t a b i l i z e d colloidal gold p a r t i c l e s t e n d to a g g r e g a t e (Figure 1). T h e y h a v e a p o l y h e d r i c a l f o r m (inset) a n d are u n s t a b l e under the electron beam. With progressive beam radiation, t h e y b e c o m e e l e c t r o n t r a n s p a r e n t (mass loss). T h e h o r s e s p l e e n ferritin (Figure 2) is q u i t e h o m o g e n e o u s ; h o w e v e r , some d e t a c h e d a p o f e r r i t i n ( p r o t e i n shell of t h e f e r r i t i n molecule) c a n be o b s e r v e d o c c a s i o n a l l y (arrow). T h e p u r i f i e d a n t i - f e r r i t i n a n t i b o d i e s (Figure 3) f o r m s m a l l clusters of fairly c o n s t a n t d i a m e t e r (30-40 nm). Aggregates of v a r i a b l e sizes of f e r r i t i n m a r k e d w i t h colloidal gold g r a n u l e s (average d i a m e t e r 5.2 n m ) are v i s u a l i z e d in F i g u r e 4. O v e r 9 5 % of t h e f e r r i t i n molecules are labelled w i t h a t least one gold particle. A d s o r p t i o n of t h e gold colloid t a k e s place o n t o t h e p r o t e i n p a r t of t h e f e r r i t i n molecules. F r e e a p o f e r r i t i n is t h e r e f o r e labelled as well. Figure 5 illustrates the purified anti-ferritin antibodies m a r k e d w i t h colloidal gold. M o s t i m p o r t a n t is t h e o b s e r v a t i o n t h a t all colloid p a r t i c l e s are c o v e r e d b y a n t i b o d i e s , i.e. a f t e r n e g a t i v e s t a i n i n g a clear, e l e c t r o n - t r a n s p a r e n t zone is visible a r o u n d all colloidal gold granules. I n f a v o u r a b l e p r o j e c t i o n s , t h e Y - s h a p e of t h e a n t i - f e r r i t i n a n t i b o d i e s a d s o r b e d o n t o t h e colloid p a r t i c l e s c a n b e r e v e a l e d (Figure 5 simple a r r o w a n d inset). I t is e v i d e n t t h a t t h i s a r r a n g e m e n t w i t h t h e colloid g r a n u l e s in t h e center, s u r r o u n d e d b y a d s o r b e d i m m u n p r o t e i n , is m o s t f a v o u r a b l e for i m m u n o c y t o c h e m i c a l r e a c t i o n s . S m a l l
clusters of free a n t i b o d i e s ( c o m p a r e w i t h F i g u r e 3) are recognized o c c a s i o n a l l y (double arrows). C o m p l e x e s b e t w e e n f e r r i t i n a n d colloidal gold, c o a t e d w i t h a n t i f e r r i t i n a n t i b o d i e s , are seen in F i g u r e 6. T h e y a p p e a r , d u e to t h e p r e s e n c e of a n t i b o d i e s , less d e n s e l y p a c k e d t h a n t h o s e f o r m e d b y f e r r i t i n a n d u n c o a t e d colloidal gold. I n t h i s p r e p a r a t i o n , r a t h e r i m p o r t a n t f l u c t u a t i o n s in t h e size of t h e colloid g r a n u l e s occurred. F i n a l l y , t h e r a t h e r heterogeneous complexes formed between ferritin marked w i t h colloidal gold a n d colloidal gold c o v e r e d w i t h a n t i f e r r i t i n a n t i b o d i e s are d e m o n s t r a t e d in F i g u r e s 7 a n d 8. W i t h a d e q u a t e c o n c e n t r a t i o n s of f e r r i t i n a n d a n t i f e r r i t i n a n t i b o d i e s , c o m p l e x e s of t h e t y p e seen in F i g u r e 7 are o b t a i n e d . All f e r r i t i n molecules are s u r r o u n d e d b y colloid particles, b u t i t is i m p o s s i b l e to d i s t i n g u i s h bet w e e n t h e gold g r a n u l e s d i r e c t l y a d s o r b e d to t h e t e r r i t i n and those linked to the ferritin via the anti-ferritin antibodies. I n F i g u r e 7, t h e f e r r i t i n a n d a n t i - f e r r i t i n a n t i b o d i e s are n o t r e v e a l e d b e c a u s e n o n e g a t i v e s t a i n i n g was applied. N e v e r t h e l e s s , t h e f e r r i t i n n u c l e u s (simple arrow) is easily d i s t i n g u i s h e d f r o m t h e gold p a r t i c l e s (double arrow) b y its c o n s i d e r a b l y lower d e n s i t y . T h e s a m e c o m p l e x e s a f t e r n e g a t i v e s t a i n i n g w i t h u r a n y l a c e t a t e are s h o w n in F i g u r e 8. A g a i n all t h e f e r r i t i n molecules are s u r r o u n d e d b y colloid granules. T h e t y p i c a l f e r r i t i n s t r u c t u r e is m a s k e d (arrow) d u e t o a d s o r p t i o n of a n t i b o d i e s . T h i s is in agreem e n t w i t h o b s e r v a t i o n s o n f e r r i t i n - a n t i - f e r r i t i n complexes 2. Discussion. F r o m t h e e l e c t r o n m i c r o g r a p h s p r e s e n t e d i t is e v i d e n t t h a t m i x e d c o m p l e x e s are v e r y h e t e r o g e n e o u s in size a n d t h e r e f o r e n o t s u i t a b l e as c y t o c h e m i c a l m a r k e r s . Two o b s e r v a t i o n s are, h o w e v e r , of i m p o r t a n c e . F i r s t l y p r o t e i n molecules are a d s o r b e d a r o u n d colloidal gold g r a n u l e s in a s t e r i c a l l y f a v o u r a b l e p o s i t i o n n e c e s s a r y for c y t o c h e m i c a l c o u p l i n g or for t h e a n t i b o d y - a n t i g e n react i o n . T h i s p r o p e r t y h a s a l r e a d y b e e n successfully e x p l o i t e d in labelling e x p e r i m e n t s in t h e t r a n s m i s s i o n -7-1~ a n d
1 A. FEINSTEIN and A. J. ROWE, Nature, Lond. 205, 147 (1965). 2 j. p. ROBINSON, J. molee. Biol. 17, 456 (1966). a j. p. ROBINSONand S. S. SCHUFFMAN,Immunology 20, 883 (1971). 4 p. SEEMAN, D. CHENG and G. H. ILES, J. Cell Biol. 56, 519 (1973). G. H. ILES, P. SEEMAN, P. NAVLOR and B. C[NADER, J. Cell Biol. 56, 528 (1973). 6 D. H. CAMPBELL,J. S. GARVEY,N. E. CREMER and D. H. SUSSDORF, in Methods in Immunology (W. A. Benjamin Inc., New York 1964), p. 131. W. P. FAULKand G. M. TAYLOR, Immunochemistry 8, 1081 (1971). 8 H. GERBER, M. HORISBERCmRand H. BAUER, Infect. hnmunity 7, 487 (1973). H. BAUER, M. HORISBERGER,D. A. BUSH and E. SIGARLAKIE, Arch. Mikrobiol. 85, 202 (1972). 10 H. BAUER,D. R. FARR and M. HORISBERGER,Arch. Microbiol. 97, 17 (1974).
All electron micrographs, except Figure 7 (unstained), are from material negatively stained with 5% UO~-acetate. The dimensions on the micrographs are given in ran. Figs. 1-3. Individual materials used in our experiments. 1. Non-stabilized gold colloid particles showing instability under the electron beam. The polyhedrical form of the individual granules is well revealed (arrow, inset), x 250,000; inset x 500,000. 2. The horse spleen ferritin preparation is quite homogeneous. Some free apoferritin is indicated by an arrow. • 250,000. 3. The anti-ferritin antibodies tend to form small clusters of fairly constant diameter ( 3 0 4 0 nm). • 250,000. Figs. 4 + 5. Direct marking of ferritin and anti-ferritin antibodies with colloidal gold. 4. The complexes formed between ferritin directly marked with colloidal gold are rather heterogeneous. Over 95% of the ferritin nmleeules are marked with at least one gold particle. • 250,000. 5. All colloid particles are coated with anti-ferritin antibodies. In favourable projections (arrow, inset), the Y-shape of the antibodies is revealed. The double arrow points to small cluster of free antibodies (eomp. Figure 3). x 250,000; inset • 500,000. Figs. 6-8. Marking of native and gold-labelled ferritin with anti-ferritin antibodies coated colloidal gold. 6. Complex formed between ferritin and colloidal gold coated with anti-ferritin antibodies. • 250,000. 7. Complexes formed between gold-labelled ferritin and colloidal gold coated with anti-ferritin antibodies. No negative staining was applied and therefore the protein part of the ferritin molecules and the anti-ferritin antibodies around the gold colloids is not revealed. Ferritin nucleus: 4; gold particles: ~ . x 250,000. 8. Complexes formed between gold-labelled ferritin and colloidal gold coated with anti-ferritin antibodies. This preparation contains an excess of free antibodies, and it appears that free antibodies attach preferentially to the ferritin molecules (arrow). • 250,000.
15.10. 1975
Specialia
r e c e n t l y in t h e s c a n n i n g - u e l e c t r o n microscope. Secondly, e v e n w i t h o u t n e g a t i v e staining, t h e gold colloid a n d t h e f e r r i t i n molecules (iron nucleus) can easily be d e t e c t e d a n d d i s t i n g u i s h e d in t h e t r a n s m i s s i o n e l e c t r o n microscope. D o u b l e labelling e x p e r i m e n t s , using colloidal gold p a r t i c l e s coated with a protein (phytohemagglutinin, antibodies,
etc.) a n d f e r r i t i n - p r o t e i n c o n j u g a t e s can t h e r e f o r e be performed simultaneously.
Summary. T h e m o r p h o l o g y of m o d e l c o m p l e x e s bet w e e n colloidal gold, ferritin a n d a n t i - f e r r i t i n a n t i b o d i e s h a s b e e n s t u d i e d in o r d e r t o e v a l u a t e t h e p o t e n t i a l of colloidal gold as a c y t o c h e m i c a l m a r k e r . I-I. BAUER, H. GERBER 12 a n d
11 M. HORISBERGER,J. Ross~r and H. BAUER,submitted for publication. 12 Pathology Institute, University of Bern, CH-3008 Bern, Switzerland. la Acknowledgment. We thank Prof. H. ISLIKER, Biochemistry Institute, University of Lausanne, for the bovine colostral antiserum against ferritin.
H y b r i d i z a t i o n of M i t o c h o n d r i a l a n d C y t o p l a s m i c Mitochondrial and Nuclear DNA
1151
M. HORISBERGER13
Research Department, Nestld Products Technical Assistance Co. Ltd., CH-1814 La Tour-de-Peilz (Switzerland), and Pathology Institute, University of Berne, CH-3008 Berne (Switzerland), 16 June 1975.
Ribosomal
T h e genetic origins of m i t o c h o n d r i a l a n d c y t o p l a s m i c r R N A s f r o m r a t liver were i n v e s t i g a t e d b y R N A - D N A h y b r i d i z a t i o n e x p e r i m e n t s . I t was s h o w n in earlier s t u d i e s ~ t h a t m i t o c h o n d r i a l r R N A s f r o m r a t or m o u s e liver were d i f f e r e n t f r o m t h e i r c y t o p l a s m i c c o u n t e r p a r t s in several of t h e i r p h y s i c o - c h e m i c a l p r o p e r t i e s . If in a d d i t i o n m i t o c h o n d r i a l r R N A s h y b r i d i z e specifically w i t h m i t o c h o n d r i a l D N A a n d n o t w i t h n u c l e a r DNA, t h i s w o u l d be an indication of a d i f f e r e n t genetic origin f r o m b o t h t y p e s of r R N A s . 2V[aterial and methods. M i t o c h o n d r i a a n d R N A were p r e p a r e d f r o m a d u l t r a t liver as d e s c r i b e d in earlier s t u d i e s 1. D u r i n g subcellular f r a c t i o n a t i o n a n d R N A e x t r a c t i o n , 2 ribonuclease i n h i b i t o r s were used to aw)id R N A d e g r a d a t i o n : a n a t u r a l one p r e p a r e d f r o m r a t liver the d a y before, a n d d i e t h y l p y r o c a r b o n a t e . A f t e r e t h a n o l p r e c i p i t a t i o n , t h e R N A s were purified b y 2 c e n t r i f u g a t i o n s t h r o u g h 5 20% sucrose g r a d i e n t s in order to completely eliminate tRNAs. T h e c y t o p l a s m i c r i b o s o m a l R N A s were p r e p a r e d a c c o r d i n g to STEVENIN e t al. 2, e x c e p t t h a t for t h e ribonuclease t r e a t m e n t , 0.05 ~*g ribonuclease per ml for 20 m i n a t 0 ~ was used to r e m o v e m R N A . M i t o c h o n d r i a l D N A was p r e p a r e d according to lx'rEL et al. a a n d t h e n u c l e a r I ) N A according to MARMUR 4, e x c e p t t h a t D N A was p r e c i p i t a t e d b y cold e t h a n o l a n d n o t b y isopropanol. B o t h D N A s were f r a g m e n t e d b y s o n i c a t i o n as d e s c r i b e d b y MORI et al. 5, in o r d e r to o b t a i n f r a g m e n t s w i t h a l e n g t h of a b o u t 500 base pairs, w h i c h c o r r e s p o n d s a p p r o x i m a t i v e l y to a gene size6, L The t w o
RNA with
species of D N A s were t r i t i a t e d in v i t r o w i t h s o d i u m a H - b o r o h y d r i d e as d e s c r i b e d b y LEE a n d GORDONS. T h e specific activities of D N A s o b t a i n e d v a r i e d f r o m 5-1500 cpm/tzg DNA. The m e l t i n g t e m p e r a t u r e s (Tms) of t h e D N A f r a g m e n t s a n d h y b r i d s were d e t e r m i n e d b y t h e r m i c elutions a t increasing t e m p e r a t u r e s on h y d r o x y a p a t i t e columns. This m e t h o d of t e m p e r a t u r e m e a s u r e m e n t s 5 is v e r y r a p i d and several d e t e r m i n a t i o n s can be d o n e s i m u l t a n e o u s l y . H y d r o x y a p a t i t e was p r e p a r e d a c c o r d i n g t o TISELIUS 9 a n d m o d i f i e d b y LEVIN ~~ T h e elution was p e r f o r m e d w i t h 0.12 M s o d i u m p h o s p h a t e buffer. Nuclease $1 was p r e p a r e d a c c o r d i n g t o SUTTON 11 in our l a b o r a t o r y f r o m t a k a d i a s t a s e . E n z y m a t i c a c t i v i t y was d e t e r m i n e d a c c o r d i n g to SUTTON n e x c e p t t h a t t h e i n c u b a t i o n was p e r f o r m e d a t 37 ~ a n d n o t a t 56 ~ This enzyme preferentially attacks single-stranded DNA. H y b r i d i z a t i o n c o n d i t i o n s were t h o s e of our l a b o r a t o r y . T r i t i a t e d D N A (12,000-15,000 cpm) was i n c u b a t e d w i t h a large excess of R N A (100 to 600 times) in o r d e r to decrease s t r o n g l y I ) N A - D N A reassociations, in 0.75 M s o d i u m p h o s p h a t e b u f f e r for 48 h a t 65~ a f t e r h e a t d e n a t u r a t i o n . H y d r o x y a p a t i t e (2 ml per column) was 1 A. I)IERICU, M. }". ITTEL and M. WINTZERITtI, l.ife Sci. 13, 553 (1973). 2 J. STEVENIN, J. SAMEC, M. JACOl~ and P. MANDEL, J. molec.
Biol. 33, 777 (1968).
a M. E. ITT~:r., M. WINTZERITH and P. MANnEL, C. r. Acad. Sci., Paris, Sdrie D 274, 3622 (1972). 4 J. MARMUR,J. molee. Biol. 3, 208 (1961}. 5 K. MORI, M. WINTZERITH and P. ~[ANDEL, Biochilnie 54, 1427
"Fable I. Hybridization percentages of mitochondrial and cytoplasmic rRNAs with mitochondrial and nuclear DNA
MitochondrialrRNA Cytoplasmic rRNA
Mitoehondrial DNA (% DNAhybridized)
Nuclear DNA (% DNA hybridized)
1.74 (10) 0.02 (3)
0.01 0.42
(3) (8)
The values in brackets are the number of detertninations.
(1972). 6 R. J. BRITTEN and D. E. KOtlNE, Science 161, 529 (1968). 7 E. H. DAVIDSONand B. R. HOUGH, Proe. natn. Aead. Sci., USA 63, 342 (1969}. s V. F. LEE and M. P. GORDON, Biochim. biophys. Aeta 2,38, 174 (1971). 9 A. TISELIUS, S. HJERTISNand O. LEVlN, Arch. Biochem. Biophys. 65, 132 (1956). 10 O. LEVIN, Methods in Enzymology (Eds. S. P. COLOWICKand N. O. KAPLAN; Acadeufie Press, New York 1962), vol. 5, p. 27. 11 W. D. SUTTON, Bioehim. biophys. Aeta 2,f0, 522 (1971).
Table ii. Temperature values frolu nuclear and mitochoudrial DNA, and RNA-DNA hybrids Temperature (~ Mitochondrial DNA Mitoehondrial DNA-mitochondrial rRNA hybrids
82 74.8
Temperature (~ Nuclear DNA Nuclear DNA-cytoplaslnic~rRNA hybrids
86 78.5
