BIOCHIMIE, 1979~ 61, 865-868.

Reactivity of anti nucleoside antibodies with mctaphase chromosomes.

J. MORIN *, M. M,ARCOIJLET ** a n d L E N G "*"

(22-3-1979).

* Laboratoire d'Histologie, Embryoiogie Cglog~ndtique, U.E.R. Mddecine, 63001 Clermont-Ferrand Cedex, France.

*'Laboratoirc de Chimie Biologique et Pharmacodynamie, (Pro[. P. Bastide) U.E.R. Pharmacie, 63001 Clermont-Ferrand Cedex, France.

" ' * Centre de Biophysiquc Mol~culaire, 1A, avenue de Ia Recherche Scientifique 450~5 Orleans Cedex, France.

Summary.

It is shown that the binding o[ anti-nucleoside antibodies to [ixed human metaphase chromo- somes can be revealed using the immunoperoxi- dase procedure while under the same conditions no antibody binding is revealed using the immuno- fluorescence procedure.

S e v e r a l s t u d i e s h a v e s h o w n t h a t i m m u n o f l u o r e s - c e n c e t e c h n i q u e s u s i n g a n t i - n u c l e o s i d e a n t i b o d i e s e l i c i t e d i n r a b b i t s c a n be u s e f u l to c h a r a c t e r i z e f ixed m e t a p h a s e c h r o m o s o m e s [ r e v i e w e d in 1]. H o w e v e r a p o s i t i v e r e a c t i o n w a s o n l y o b s e r v e d a f t e r p a r t i a l d e n . a t u r a t i o n of t h e DN,A ( fo r e x a m p l e b y u l t r a v i o l e t i r r a d i a t i o n , b y t r e a t m e n t w i t h for - m a m i d e at h i g h t e m p e r a t u r e , o r b y p h o t o - o x i d a - t i o n of g u a n i n e r e s i d u e [2-5t). On t h e o t h e r h a n d , i t w a s o f t e n n o t e d t h a t t h e use of a c e t i c a c i d a n d a l c o h o l as cel l f i xa t i ve s c a n p e r t u r b t he c h r o m o - s o m e s [ r e v i e w e d in 6]. V e r y r e c e n t l y , i t w a s r e p o r t e d t h a t a c e t i c a c i d t r e a t m e n t of c h r o m o - s o m e s c a n r e s u l t i n DN~A d e n a t u r a t i o n w h i l e t h e m o r p h o l o g y of t h e c h r o m o s o m e s is p r e s e r v e d [6].

I n g e n e r a l , i m m u n o l o g i c a l m e t h o d s a r e v e r y s e n s i t i v e a n d t h u s i t s e e m e d to us of i n t e r e s t to s t u d y w h y n o r e a c t i o n w a s f o u n d b e t w e e n t he a n t i n u c l e o s i d e a n t i b o d i e s a n d c h r o m o s o m e s w h i c h h a v e n o t b e e n t r e a t e d b y p h y s i c a l or c h e m i c a l d e n a t u r i n g r e a g e n t s . I n t h i s w o r k , w e r e p o r t s o m e o b s e r v a t i o n s b y l i g h t m i c r o s c o p y o n t h e

b i n d i n g of h i g h l y pu,r i f led a n t i a d e n o s i n e a n t i - b o d i e s a n d of a n t i c y t i d i n e a n t i b o d i e s t o h u m a n c h r o m o s o m e s , t h e r e g i o n s of a n t i b o d y b i n d i n g b e i n g v i s u a l i z e d ~by t h e use of a n i m m u n o - p e r o x i - d a s e p r o c e d u r e a n d of a n i m m u n o f l u o r e s c e n e e

p r o c e d u r e .

Material and Methods.

Chromosome p repa ra t ions were made f rom h u m a n lymphoeytes cul tures [7]- Metaphase chromosomes spreads were prepared by adding colchicine to the me- d ium at a final concent ra t ion of 1 × 10-4 g/1. After the hypotonica l t rea tment , the cells were fixed in a mix tu re of acetic acid, e thanol , ch loroform (10:60:30 and 15:75:10, v / v / v respectively) and then were dried. The slides were used wi th in two days.

The slides were u l t r av io le t i r r ad ia ted dur ing 18 hours w i th a 30 W germicidal l amp (General Electric) as described [5]. The pur i f ica t ion of the an t i -adenos ine an t ibodies has been a l ready repor ted [8]. A s imi la r procedure was used to pur i fy the an t i -cy t id ine an t i - bodies.

An t i - r abb i t IgG an t ibodies label led wi th fluorescein (Lot N ° 70314) and an t i - r abb i t IgG ant ibodies label led w i th peroxidase (Lots N ° 04 75 011 and 06 75 011) were purchased f rom the Ins t i tu t Pasteur .

The fol lowing procedure was used to label the chro- mosomes wi th the an t ibodies [2, 9] : the slides were r insed in s tandard phospha te buffered sal ine (PBS buffer) and then layered wi th the ant i -nucleos ide an t i - bodies (0.5 ml of a solut ion 0.1 m g / m l in PBS).

After 30 rain in a humid chamber at 37°C, they were r insed wi th a spray of PBS buffer, immersed twice dur ing ten minu te s in PBS buffer and then layered w i th the fluoreseein label led ant ibodies (0.5 ml of a solut ion 0.034 mg/ml ) or wi th the peroxidase label led an t ibodies (0.5 ml of a solut ion 0.0125 mg/ml ) . After

60

8 6 6 J . M o r i n a n d c o l l .

30 min at 37°C in a h u m i d chamber the slides label led wi th the fluorescein ant ibodies were r insed wi th a spray of dis t i l led wate r immersed twice in dis t i l led wate r and a i r dried in the dark. The slides label led w i th the peroxidase ant ibodies were immersed for ten minu tes in a solut ion (0.075 g 3 ,3 ' -diaminobenzidine,

w i t h t h e u l t r a v i o l e t t r e a t e d c h r o m o s o m e s t h a n w i t h fhe u, n t r e a t e d c h r o m o s o m e s ( f igure 2). H o w e - ver , t h e u n t r e a t e d c ' h r o m o s o m e s d i d r e a c t w i t h t h e a n t i b o d i e s . Also, t h e s t a i n i n g w a s d i f f e r e n t w i t h t h e u n t r e a t e d c h r o m o s o m e s a n d t h e a n t i - c y t i d i n e

FIG. 1. - - U l t rav i o l e t i r rad ia t ed h u m a n c h r o m o s o m e s s t a ined w i t h a n t i - a d e n o s i n e an t i bod i e s by an ind i rec t i m m u n o f l u o r e s c e n c e proce- dure .

3 × 10-5 ml H~O~ a t 30 per cent, 100 ml H~O). They were then r insed wi th PBS, passed th rough an alcohol series (40-100 per cent) and finally a i r dried.

A Dialux 20 Leitz microscop, equipped wi th fluores- cence was used (100 × Phaco 3 objective). The photo- graphs were taken on microfi lms Kodak and Kodak plus X film.

R e s u l t s a n d D i s c u s s i o n .

T h e r e s u l t s a re p r e s e n t e d in f i gu re s 1 a n d 2. A f t e r u l t r a v i o l e t i r r a d i a t i o n , t he , b ind ing of a n t i - a d e n o s i n e a n t i b o d i e s ( f igure 1) a n d of a n t i - c y t i - d i n e a n t i , b o d i e s ( r e su l t s no t s h o w n ) to c h r o m o - s o m e s w a s ea s i ly f o l l o w e d b y f l u o r e s c e n c e . No f l u o r e s c e n c e w a s o b s e r v e d w h e n t 'he c h r o m o s o m e s w e r e n o t t r e a t e d w i t h u l t r a v i o l e t l igh t . On t h e o t h e r h a n d , t h e b i n d i n g of t he a n t i a d e n o s i n e a n t i - b o d i e s a n d of the a n t i c y t i d i n e a n t i b o d i e s to c h r o - m o s o m e s w a s f o l l o w e d b y t he p e r o x i d a s e p r o c e - d u r e . Q u a l i t a t i v e l y , t h e s t a i n i n g w a s m o r e i n t e n s e

BIOCHIMIE, 1979, 61, n o 7.

a n t i b o d i e s o r t h e a n t i - a d e n o s i n e a n t i b o d i e s b u t in b o t h eases a p o s i t i v e r e a c t i o n w a s f o u n d .

To b e s u r e t h a t w e w e r e d e a l i n g w i t h spec i f i c i n t e r a c t i o n s , s e v e r a l e x p e r i m e n t s w e r e d o n e u s i n g t h e p e r o x i d a s e p r o c e d u r e w i t h t h e f o l l o w i n g m o d i - f i c a t i o n s :

- - T h e a n t i - a d e n o s i n e a n t i b o d i e s w e r e n o t a d d e d or w e r e r e p l a c e d b y n o n - s p e c i f i c r a b b i t IgG. No s t a i n i n g w a s f o u n d .

- - A s o l u t i o n of a d e n o s i n e (1 p e r cen t ) w a s l a y e r e d b e f o r e t h e a d d i t i o n o~f t h e a n t i - a d e n o s i . n e a n t i b o d i e s . No s t a i n i n g w a s o b s e r v e d .

- - T h e s t a i n i n g w a s less i n t e n s e b u t s t i l l de t ec - t a b l e w h e n a d i l u t e s o l u t i o n of a n t i - a d e n o s i n e a n t i - b o d i e s (0.1~1' r n g / m l ) w a s u s e d or w h e n t h e a n t i - b o d i e s -~'ere d i s s o l v e d i n 1 M NaC1 F H 7 i n s t e a d

of P B S bu f fe r .

- - Af te r t h e r e a c t i o n w i t h t h e a n t i - a d e n o s i n e a n t i b o d i e s , t h e s l i d e s m e r e w a s h e d witch 1 M NaC1

M e t a p h a s e c h r o m o s o m e s . 867

instead of PB, S ~buffer. Quali tat ively the same results were found.

Our results us ing the immunofluorescence pro- cedure are in agreement wi th those already repor- ted [1-4] but in ad'dition using the peroxidase procedure, we show that the anti adenosine anti- bodies and the anti cyt idine ant ibodies b ind to

and chromosomes is unl ikely. This was also con- firmed by wash ing the slides wi th a concent ra ted salt solution (1 M l'~aC1).

The react ion between the ant i -adenosine anti- bodies and adenosine residues in DN~A is possible only if the adenine residues are accessible (not pai red with thymine residues [1, 8]. Our results

_ s = : $ ~ t$ t)

2 . ~ 2"b

kg'

p 2.c 2 .d

FIG. 2 . - Human chromosomes stained with anti-cytidine antibodies (a, b) and anti-adenosine antibodies (e, d) by an indirect immunoperoxidase procedure. In b and d the chromo- somes were ultraviolet irradiated.

chromosomes wh ich were not treated with a phy- sical or chemical denatt trat ion reagent.

The react ion is due to a specific b ind ing of the purif ied ant i -nucleoside antibodies. The addi t ion of adenosine prevents the b ind ing of the anti- adenosine ant ibodies to DN~A. The non specific rabbi t IgG do not b ind to ,chromosomes in 0.2 M NaC1. From this result a non-specific electrostatic in te rac t ion between the anti adenosine ant ibodies

BIOCHIMIE, 1979, 61, n ° 7.

show that some regions of the DNA are denatured. It is not necessary to be in the presence of long single s t randed regions. We found [8] that each b ind ing site of the ant ibodies to adenosine covers about 3 adenosine residues in oligo (A) (it should be noted that the size of the b ind ing site of the ant ibodies is ra ther small ; we have also reported that each b ind ing site of the ant ibodies to the double-s t randed polynucleot ide poly ,(D.poly (C)

868 J. M o r i n a n d coi l .

covers 3, base pa i r s [10] ). The ant ibodies can react w i th small loops in DN'A and therefore can be more sensi t ive than wi th other techniques for de tec t ing short s ingle-s t randed regions.

We do not k n o w xvhether the loops are present in unfixed Chromosomes or are due to the treat- ment used to fix the chromosomes. We can only say that the lat ter hypothesis seems more l ikely. On the other hand, we have shown that the anti- nucleoside(t ide) ant ibodies and the anti-single- s t randed polynucleo t ide ant ibodies can behave as tmvcinding pro te ins in the presence of double- s t randed polynucleot ides [8, I1, 12]. Thus it is also possible that some ant ibodies react wi th some unstable regions.

In conclusion, because of the h igher sensi t iv i ty of the immunope rox idase p rocedure as c o m p a r e d to the immunof luoreseence procedure , some dena- tured regions in fixed human chromosomes have been detected.

This work was supported in part by the Ddl~gation G~ndrale ~ la Recherche Scientifique el Technique (contract n ° 79 7 0151).

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6. Shapiro, I. M., Moar, M. H., Ohno, S. ~ Klein, G. (1978) Exp. Cell. Res., 115, 411-414.

7. Dutrillaux, R., Th6se, Paris, 1975. 8. Lavayre, J. ~ Leng, M. (1977) Biochimie, 59, 33-42. 9. Lubit, B. W., Pham, T. D., Miller, O. J. • Erlanger,

B. F. (1976) Cell, 9, 503-510. 10. Leng, M., Gnigues, M. ~ Genest, D. (1978) Bioche-

mistry, 17, 3215-3220. 11. Sage, E. & Leng, M. (1977) Biochejnistry, 16, 4253-

4257. 12. Guigues, M. ~ Leng, M. (1976) Eur. J. Biochem.,

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BIOCHIMIE, 1979, 61, n" 7.