bernard malfoy, brighte hartmann and marc leng centre de

volume 9 Number 211981 Nucleic Acids Research

The B-»Z transition of poly(dG-dC).poly(dG-dC) modified by some platinum derivatives

Bernard Malfoy, Brighte Hartmann and Marc Leng

Centre de Biophysique Moleculaire, C.N.R.S., 1A, avenue de la Recherche Scientifique, 45045Orleans Cedex, France

Received 25 September 1981

SUMMARY

Poly(dG-dC).poly(dG-dC) was modified by chlorodiethylenetriamino platinum(II) chloride, cis-dichlorodiammine platinum (II) and trans-dichlorodiammineplatinum (II), respectively. The conformation of these modified poly(dG-dC).poly(dG-dC) was studied by circular dichroism. In 4 M Na+, the circular di-chroism spectra of poly(dG-dC)<iiew-Pt (0 < rj, « 0.2) are similar (rb is theamount of bound platinum per base). It is concluded that the conformation ofthese polymers belongs to the Z-family. Dien-Pt complexes stabilize the Z-form. The midpoint of the Z ->• B transition of poly(dG-dC)dien-Pt(0.12) is at0.2 M NaCl. Moreover another B •* Z transition is observed at lower salt con-centration (midpoint at 6 mM NaCl). In 1 mM phosphate buffer, the stabilityof Z-poly(dG-dC)dien-Pt(0.12) is greatly affected by the presence of smallamounts of EDTA. Poly(dG-dC).poly(dG-dC) modified by ais-Pt and trans-Pt com-plexes do not adopt the Z-form even in high salt concentration.

The therapeutic efficiency of cis-dichlorodiammine platinum (II) (eis-Pt)

on tumor is now well-established. It has been demonstrated that cis-Pt binds

to DNA and several lines of evidence suggest that this binding is related to

anti-tumor activity of this compound (general reviews, 1,2 and references

herein). Numerous studies have been carried out in an attempt to describe the

modification of DNA. In vitro, ais-Pt compound binds strongly to DNA and gua-

nine residues are the most preferred binding site (1,3,4). The recent discove-

ry that oligo(dG-dC) crystals (5-7) and poly(dG-dC).poly(dG-dC) fibers (8) can

adopt the Z-form lead us to ask the question whether the binding of cis-Pt to

poly(dG-dC) .poly(dG-dC) could hinder or favour the B •+ Z transition. It has

been already shown that the covalent binding of some products to guanine resi-

dues in poly(dG-dC).poly(dG-dC) favours the Z-form (9-12). Moreover, as alrea-

dy done by other investigators, it seemed to us of interest to compare the ef-

fects of two other platinum compounds, trans-dichlorodiammine platinum (II)

(trans-Pt) and chlorodiethylenetriamino platinum (II) chloride (dien-Pt) which

have no anti-tumor activity.

In this paper, we report some results obtained by circular dichroism on

poly(dG-dC).poly(dG-dC) modified by these three platinum compounds. It is

© IRL Press Umited. 1 Falconberg Court. London W1V SFG. U.K.

Downloaded from https://academic.oup.com/nar/article-abstract/9/21/5659/2380013by gueston 19 February 2018

Nucleic Acids Research

known that in solution poly(dG-dC).poly(dG-dC) can undergo a reversible salt

induced conformation change with a midpoint at about 0.7 MgCl_ or 2.5 M NaCl

(13). The low-salt form was shown to belong to the B-family (14) and the high-

salt form to the Z-family (13,15-18). Circular dichroism is very convenient to

study these two forms. The circular dichroism spectrum of the high-salt form

is almost an inversion of the spectrum of the low-salt form (13,19). In this

work, as judged by circular dichroism, we show that cis-Pt and trans-Pt com-

plexes prevent the transition of poly(dG-dC).poly(dG-dC) to the Z-form. On the

other hand, we found that poly(dG-dC).poly(dG-dC) modified by dien-Vt compound

undergoes two transitions from the B-form to the Z-form or Z-like form and

that these two transitions occur at much lower salt concentrations than that

of unmodified poly(dG-dC) .poly(dG-dC) .

MATERIAL AND METHODS

Poly(dG-dC).poly(dG-dC) bought from P.L. Biochemicals was treated with

phenol and then exhaustively dialyzed as already described (11). Platinum com-

pounds were a gift of Dr. J.P. Macquet (Toulouse). The quantitative fixation

of platinum compounds on poly(dG-dC).poly(dG-dC) was performed as described by

Macquet and Butour (20). The platinum compounds were dissolved just before

used in 10 mM NaClO^ and added to a 200 ug/ml solution of poly(dG-dC).poly(dG-

dC) in the same medium. The reaction was run at 37°C in the dark for 24 hours.

The modified samples were exhaustively dialyzed against 1 mM phosphate buffer,

pH 7.3. The platinum content of some samples was determined with an atomic

absorption spectrometer (21). We will call r, the number of platinum atoms

bound per nucleotide. We will write poly(dG-dC)cis-Pt(0.15), poly(dG-dC)trans-

Pt(0.15) and poly(dG-dC)diew-Pt(O.15) a sample of poly(dG-dC).poly(dG-dC) com-

plexed respectively with eis-Pt, trans-Pt and dien-Vt at rb of 0.15.

Methylation of guanine residues of poly(dG-dC).poly(dG-dC) was performed

as already described (22). Dimethylsulfate (Aldrich) (total volume 6 yl) was

added in three times every 30 minutes to a solution of poly(dG-dC).poly(dG-dC)

(0.5 mg in 1 ml of 0.5 M sodium cacodylate, pH 7). The product was then exhaus-

tively dialyzed against 1 mM phosphate buffer. The percentage of modified ba-

ses was determined by gel filtration chromatography on Sephadex G-10 after

acid hydrolysis of the polymer (23) .

Ultraviolet absorption (UV) and circular dichroism (CD) spectra were re-

corded with a Cary 210 spectrophotometer and a Roussel Jouan III dichrograph,

respectively.

5660



RESULTS

The formula of the three compounds which were respectively reacted with

poly(dG-dC).poly(dG-dC) are the following :

H3N

\

Cl

Ptt

\H3N Cl

H3N Cl

\ /Pt

/ \Cl NH3

cis-Pt(NH3)2CI2 trans-Pt(NH3)2CI2

H2C_CH2

HN NH2

H2N Cl

[pt(dien)Cl]ci

Dien-Pt compound has only one reactive function while cis-Pt and tvans-

Pt have two.

1) Dien-Pt compound

In figure 1, we compare the CD spectra of poly(dG-dC).poly(dG-dC) and of

poly(dG-dC)<f£en-Pt at various r,, in 1 mM phosphate buffer.

The CD spectrum of poly(dG-dC).poly(dG-dC) presents a positive band and

A£

4

2

-2

-4

2

- -2

-<

^ 2 9 0

0.05 0.1

•

A

0.15 rb £?"%•

<\ / / ?.\ • • • • • ' / /

\ /v. iV

•+'•••"+/

+ +/

>-'

B

/ \/ \

. — - '

-

At

8

6

2

-2

-6

250 300 250WAVELENGTH nm

300

Fig. 1 - Circular dichroism spectra. Poly(dG-dC)dien-Pt at various rD. Medium1 mM phosphate buffer pH 7.3. A) rb = 0 ; — rb = 0.05 ; ••• r0 = 0.08 ;

rjj = 0.12 ; +++ r^ = 0.22. Inset : variation of Ae290nm a s a function ofr b . B) +++ r b = 0.22, r b = 0 . 2 5 ; r b = 0.30.

5661



then a negative one as already reported in the literature (19). The binding

of dien-Pt compound to poly(dG-dC).poly(dG-dC) modified greatly the CD spec-

trum. As rb increases, the first positive band decreases and then becomes ne-

gative while the negative band becomes positive. At rj, = 0.12 the spectrum of

poly(dG-dC)dien-Pt(0.12) is almost an inversion of the spectrum of poly(dG-

dC).poly(dG-dC) . The shape of the spectra is almost the same for 0.12 < r^ <

0.2. At larger values of r,, new changes were observed in the spectra. At r^=

0.3, the spectrum presents an intense positive band centered at 295 nm and

then a negative band (figure IB).

As shown in the inset of figure 1A, the largest absolute value of Ae.gg

was found at r. = 0.12. This modified sample was studied in more detail.

The CD spectra of poly (dG-dC)cKerc-Pt(O.12) recorded in various media are

shown in figure 2. In 4 M NaCl (or NaClO^) the CD spectra of poly(dG-dC)dt:en-

Pt(0. 12) and of poly(dG-dC).poly(dG-dC) are identical (for sake of clarity,

the spectrum of poly(dG-dC).poly(dG-dC) is not shown). In 1 mM phosphate buf-

fer, the spectrum of poly(dG-dC)<2ien-Pt(0. 12) is slightly different from that

in 4 M NaCl. The first negative bands are identical but the positive band of

-4

-6

250 300WAVELENGTH nm

Fig. 2 - Circular dichroism spectra of poly(dG-dC)dien-Pt(0.12) as a functionof salt concentration. NaCl concentration : 0.1 mM ; ••• 5 mM ; 30mM ; +++ 100 mM ; —•— 1 M and 4 M. All the solutions contain 1 mM phosphatebuffer pH 7.3. Inset : variation of ^290 a s a function of the logarithm ofsodium concentration.

5662



the spectrum in 1 mM phosphate buffer is less intense and red-shifted. In 30

mM NaCl the spectrum is completely different, the first band being positive

and the second one negative.

The transition between these different forms are cooperative. This is

shown in the inset of figure 2 where the variation of A£2go (other wave-

lengths can be used) is plotted as a function of NaCl concentration. The mid-

point of the first transition is at about 6 mM NaCl and of the second transi-

tion at 0.2 M NaCl.

Addition of EDTA destabilizes this low-salt form of poly(dG-dC)dien-Pt

(0.12). The CD spectrum of poly(dG-dC)die«-Pt(0.12) in 1 mM phosphate buffer

plus 0.1 mM EDTA is identical to that in 30 mM NaCl (positive band and then

negative band).

2) Cis-Pt compound

The CD spectra of two poly(dG-dC)cis-Pt, in 1 mM phosphate buffer, are

shown in figure 3A. The spectrum of poly(dG-dC)eis-Pt(O.15) presents a small

negative band at 295 nm, then a positive band centered at 275 nm and then a

positive band at 225 nm. Even at higher r, , there was no inversion of the CD

spectrum. All the experiments were done within two days after the modification.

Some evolutions in the CD spectra were observed as a function of time. After a

few days, the first negative band became smaller and the first positive band

A£4

2

-2

-i.

. A . B

250 300 250WAVELENGTH nm

300

Fig. 3 - Circular dichroism spectra. A) poly(dG-dC)eie-Pt, B) poly(dG-dC)irans-Pt ; rb = 0 ; rD = 0.05 ; —•— rj, - 0.15. Medium 1 mM phosphate buf-fer, pH 7.3.

5663



became larger ( resu l t s not shown).

3) Trans-Pt compound

The CD spectrum of poly(dG-dC)ir>ans-Pt(0.15) differs from the spectrum

of poly(dG-dC).poly(dG-dC) but the changes are not so drastic as those found

with dien-Ft and eis-Pt complexes (figure 3B). The first positive band is red-

shifted and the intensity of the negative band is smaller with a small red-

shift of the minimum. Changes in the spectra were also observed as a function

of time.

Finally we have studied poly (dG-dC).poly(dG-dC) modified by the three

platinum compounds respectively in 4 M NaCIO,. The spectra of poly(dG-dC)dien-

Pt(0.12), poly(dG-dC)cis-Pt(0.15) and poly(dG-dC)ferans-Pt(0.15) are shown in

figure 4. The spectra of poly(dG-dC).poly(dG-dC) and poly(dG-dC)dien-Pt(0.12)

are identical and very different from those of poly(dG-dC)eis-Pt(0.15) and of

poly(dG-dC)fcrons-Pt(0. 15) . Similar results were obtained in 4 M NaCl.

4) Methylated poly(dG-dC).poly(dG-dC) and poly(dG-dC).poly(dG-dC) in ethanol

In order to explain some of our results (see discussion) we have studied

on one hand a methylated poly(dG-dC).poly(dG-dC) and on the other hand poly

(dG-dC).poly(dG-dC) in presence of ethanol.

Poly (dG-dC).poly(dG-dC) was reacted with dimethylsulfate and about 30 %

of the guanine residues (on the N(7)) were modified. The CD spectra in 1 mM

A t

3

1

-1

-3

»* • * *

N • s * \

7 • *\ \

250 300

WAVELENGTH nm

Fig. 4 - Circular dichroism spectra. •• • poly(dG-dC)dien-Pt(0.12), poly(dG-dC)trans-Pt(0.15 ; poly(dG-dC)cis-Pt(0.15). Medium 4 M NaClO^ plus1 mM phosphate buffer, pH 7.3.

5664



phosphate buffer and 4 M NaCl are shown in figure 5. The spectrum in 4 M NaCl

looks like the spectrum of poly(dG-dC).poly(dG-dC) in the same solvent. The

low salt form •*• high salt form transition is cooperative and the midpoint is

about at 2 M NaCl (inset, figure 5).

The stability of poly(dG-dC).poly(dG-dC) in presence of ethanol was also

studied by CD. It has been already shown that addition of alcohol induces the

B ->• Z transition (19). In 1 mM phosphate buffer, 0.1 mM EDTA results similar

to those already published (19) were found. The midpoint of the transition is

at 60 % ethanol. The experiment was repeated in the same buffer without EDTA.

The midpoint of the transition is at about 35 % ethanol (results not shown).

DISCUSSION

In this work we compared the conformation of poly(dG-dC).poly(dG-dC) mo-

dified respectively by the two bifunctional compounds cis-Pt and trans-Pt and

by the monofunctional compound dien-Pt. These compounds bind strongly to poly

(dG-dC).poly(dG-dC) but their effects are very different.

It has been found that guanosine-5'-monophosphate and cytidine 5'-mono-

phosphate coordinate to dien-Pt through N(7) and N(3) respectively (24). In

4

2

-2

-4

At 290

/ \ "1

'. -3' i

1 -5

/

= I-3 -2 -1 0

lo,[Na*]

WS /

250 300WAVELENGTH, nm

Fig. 5 - Circular dichroism spectra. Methylated poly(dG-dC).poly(dG-dC) (0.3).1 mM phosphate buffer pH 7.3 ; 4 M NaCl plus 1 mM phosphate buffer

pH 7.3. Inset : variation of Ae290nm a s a func ti°n °f t n e logarithm of sodium

ticoncentration.

5665



double stranded helices, the N(3) of cytosine residues are not accessible and

thus are not expected to react. This has been verified on poly(I).poly(C) (25,

26). In the reaction between nucleic acids and cis or irons-platinum compounds

the N(7) of guanine residues are probably involved (1,2). However, bidendate

complexes can be formed and it is not yet known with certainty whether one or

two nucleotide residues react.

Let us discuss first the results obtained on poly(dG-dC).poly(dG-dC) modi-

fied by dien-Pt compound.

In 4 M NaCl (or NaClO^) the shape of the CD spectra of poly(dG-dC)dien-Pt

(0 ̂ r, < 0.20) are similar. These spectra have a first negative band centered

at 292 nm and then a positive band centered at 262 nm. They are almost an in-

version of the CD spectrum of poly(dG-dC).poly(dG-dC) in 1 mM phosphate buffer.

Several results show that the high salt conformation of poly(dG-dC).poly(dG-

dC) belongs to the Z-family (13,15-18) and thus we conclude that poly(dG-dC)

dierz-Pt (0 < rfe < 0.20) in 4 M Na adopts the Z-form.

Dien-Pt complexes stabilize the left-handed helix. The midpoint of the

B •+ Z transition for poly(dG-dC)dierc-Pt(0.12) is at about 0.2 M NaCl (2.5 M

NaCl for poly(dG-dC).poly(dG-dC) (13)). We can only speculate on the mechanism

by which dien-Pt complexes increase the relative stability of the left-handed

helix. One can assume an alteration in dipole moment and polarizability due

to the bound dien-Pt, a charge effect (modified guanine residues bear two po-

sitive charges), a steric hindrance in the B form, the existence of hydrogen

bonds between the NH- or NH groups of the dien-Pt and the adjacent nucleotides

or between a phosphate group and the hydrated dien-Pt in the Z-form and not

in the B-form. All these factors can play a role.

The dten-Pt residues are bulky and,as already noted (5), the N(7) of gua-

nine residues are more accessible in Z-form than in B-form. On the other hand,

the conformation of DNA is hardly altered by the binding of dien-Pt residues

as judged by CD (20,27).

It has been already reported that dien-Pt complexes with poly(I).poly(C)

are thermally more stable than poly(I).poly(C). It was proposed (26) that hy-

drogen bonds are formed between the amine groups of the dien-Pt residues

and the adjacent bases. Hydrogen bonds might be more efficient in Z-form than

in B-form of poly(dG-dC)dien-Pt.

Modifications of guanine residues by mitomycin (9) or by acetylamino-

fluorene residues (10-12) stabilize the Z-form. The covalent binding of ace-

tylaminofluorene residues to the C(8) of guanine residues favours the syn con-

formation of the modified nucleotides (28,29) which can stabilize the Z-form

5666



(in the Z-fonn, the guanine residues have the syn conformation (5)). It is not

yet known whether the binding of dien-Pt to the N(7) of guanine residues can

favour the syn conformation.

To look at the charge effect we compared poly(dG-dC).poly(dG-dC) and a

poly(dG-dC).poly(dG-dC) sample in which about 30 % of the bases were methyla-

ted (on the N(7)). There was no change in the CD spectrum of the methylated

sample in the range 1 mM-1.5 M salt. An inversion of the CD spectrum was ob-

served at higher ionic strength. The midpoint of the transition is at 2 M NaCl

(2.5 M NaCl for poly(dG-dC).poly(dG-dC)). The methylated sample and poly(dG-

dC)dien-Pt(0.12) have about the same number of positive charges. However, each

modified guanine residue bears two positive charges in poly(dG-dC)<£ien-Pt as

compared to one in the methylated poly(dG-dC).poly(dG-dC). This might explain

the larger effect of dien-Vt.

The CD spectra of poly(dG-dC)dten-Pt(O.12) and of poly(dG-dC).poly(dG-dC)

in high salt concentration are very similar and we concluded that the two poly-

nucleotides have the same conformation. This conclusion has been confirmed by

immunochemical studies (30 and work to be published). In 0.1 M NaCl, 1 mM

MgCl2 the antiserum of a rabbit immunized with poly(dG-dC)dien-Pt(0.12) elec-

trostatically bound to methylated bovine serum albumin, precipitates poly(dG-

dC)d£en-Pt(0.12) and does not precipitate poly(dG-dC).poly(dG-dC). At high

salt concentration, poly(dG-dC).poly(dG-dC) and poly(dG-dC)dien-Pt(0.12) are

precipitated by the antiserum.

The midpoint of Z + B transition of poly(dG-dC)<i£e?3-Pt(0.12) is at about

0.2 M NaCl. As the NaCl concentration was still decreased, there was a new

cooperative transition (midpoint at 6 mM NaCl). The CD spectrum of poly(dG-dC)

dien-Pt(0.12) in 1 mM phosphate buffer looks like that in 4 M NaCl. However,

the positive band is less intense and red-shifted (figure 2). Because of the

similarity between the spectra in low (1 mM) and high (4 M) ionic strength,

we assume that in 1 mM phosphate buffer we are still dealing with a left-

handed helix, the geometry of this helix being slightly different from that in

4 M NaCl (or NaC104).

The stability of this low-salt form is decreased by addition of EDTA.

Wang et at. (31) have described a Zj.-form in oligo(dC-dG) crystals. In this

Z .-form, some phosphate groups form hydrogen bonds to hydrated magnesium ions

complexed to the N. of guanine residues. It is tempting to speculate that in

the low-salt form of poly(dG-dC)dten-Pt(0.12), the dien-Yt residues bound to

the N7 of guanine residues form a bridge through water to close phosphodiester

oxygens. This would be a major stabilizing factor which could be destroyed by

5667



EDTA and NaCl.

It is not possible to verify by immunochemical experiments that in 1 mM

phosphate buffer a Z-form is observed. In this low ionic strength, non speci-

fic interactions occur between the antiserum and double stranded polynucleo-

tides.

The B •* Z transition of poly(dG-dC) .poly(dG-dC) is also affected by the

presence of small amounts of EDTA. The midpoint of the B •* Z transition indu-

ced by addition of ethanol is at about 60 % in presence of 0.1 mM EDTA (in

agreement with the literature (19)) and at 35 % in absence of EDTA. We have

previously reported that in 1 mM phosphate buffer, the percentage of Z-form in

poly(dG-dC).poly(dG-dC) modified by acetylaminofluorene residues is dependent

upon the presence of EDTA (11). These results can be understood assuming that

small amounts of multivalent ions can stabilize a Z-form (traces of multiva-

lent ions are always present despite the use of twice distillated water and

first grade reagents). This is also in agreement with the results of Behe and

Felsenfeld (32) who found that some multivalent ions are very efficient to

induce the Z-form in poly(dG-m dC).poly(dG-m dC).

As more dien-Vt complexes are bound to poly(dG-dC).poly(dG-dC) (r, > 0.2),

a new transition occurs. The CD spectrum of poly(dG-dC)dien-Pt(0.3) has an in-

tense positive band centered at 295 nm and then a large negative band. Work is

in progress to elucidate this new conformation.

Cis and trans platinum complexes do not seem to induce the Z-form. The

analysis of the results is difficult because there are some changes in the

spectra as a function of time (it should be noted that no changes were obser-

ved with poly(dG-dC)dien-Pt). In 1 mM phosphate buffer, poly(dG-dC).poly(dG-

dC) modified by cis and trans platinum compounds are not in the Z-form. More-

over, even in 4 M Na+, poly(dG-dC)c?:s-Pt(0. 15) and poly(dG-dC)trans-Pt(O.15)

do not adopt the Z-form. Cis and trans platinum complexes prevent the B •+ Z

transition of poly(dG-dC).poly(dG-dC) probably by inter or intrastrands cross-

links.

ACKNOWLEDGEMENTS

We thank Professor C. Helene for his interest in this work. The comments

of Dr. W. Guschlbauer, Dr. J. Ramstein and Dr. E. Sage are appreciated. We are

indebted to Dr. J.P. Macquet (Toulouse) for the gift of platinum compounds.

This work was supported in part by Delegation Ggngrale 3 la Recherche Scienti-

fique et Technique, contract n° 79-7-0064.

5668



REFERENCES

1. Kleinwachter, V. (1978) Studia Biophysica 73, 1-17.2. Roberts, J.J. and Thomson, A.J. (1979) in Progress in Nucleic Acids Re-

search and Molecular Biology, Davidson, I.N. and Cohn, W.E., eds, vol. 22,pp. 71-133, Academic Press, New-York.

3. Stone, P.J., Kelman, A.D. and Sinex, F.M. (1974) Nature (London) 251, 736-737.

4. Stone, P.J., Kelman, A.D., Sinex, F.M., Bhargava, M. and Halvorson, H.D.(1976) J. Mol. Biol. 104, 793-801.

5. Wang, A.H.J., Quigley, G.J., Kolpak, F.J., Crawford, J.L., Van Boom, J.H.,Van der Marel, G. and Rich, A. (1979) Nature (London) 282, 680-686.

6. Crawford, J.L., Kolpak, F.J., Wang, A.H.J., Quigley, G.J., Van Boom, J.H.,Van der Marel, G. and Rich, A. (1980) Proc. Nat. Acad. Sci. USA 77, 4016-4020.

7. Drew, H., Takano, T., Takano, S., Itakura, K. and Dickerson, R.E. (1980)Nature 286, 567-573.

8. Arnott, S., Chandrasekaran, R., Birdsall, D.L., Leslie, A.G.W. andRatcliff, R.L. (1980) Nature (London) 283, 743-745.

9. Mercado, CM. and Tomasz, M. (1977) Biochemistry 16, 2040-2046.10. Sage, E. and Leng, M. (1980) Proc. Nat. Acad. Sci. USA 77, 4597-4601.11. Sage, E. and Leng, M. (1981) Nucleic Acids Res. 9, 1241-1250.12. Santella, R.M., Grunberger, D., Weinstein, B., Rich, A. (1981) Proc. Nat.

Acad. Sci. USA 78, 1451-1455.13. Pohl, F.M. and Jovin, R.M. (1972) J. Mol. Biol. 67, 375-396.14. Pohl, F.M., Ranade, A. and Stockburger, M. (1974) Biochim. Biophys. Acta

385, 85-92.15. Patel, D.J., Canuel, L.L. and Pohl, F.M. (1979) Proc. Nat. Acad. Sci. USA

76, 2508-2511.16. Ramstein, J. and Leng, M. (1980) Nature (London) 288, 413-414.17. Mitra, C.K., Sarma, M.H. and Sarma, R.H. (1981) Biochemistry 20, 2036-

2041.18. Pilet, J. and Leng, M. (1981) Proc. Nat. Acad. Sci. USA, in press.19. Pohl, F.M. (1976) Nature (London) 260, 365-366.20. Macquet, J.P. and Butour, J.L. (1978) Biochimie 60, 901-914.21. Macquet, J.P., Hubert, J. and Theophanides, T. (1974) Anal. Chim. Acta

72, 251-259.22. Ramstein, J., Helene, C. and Leng, M. (1971) Eur. J. Biochem. 21, 125-136.23. Goth, R. and Rajewsky, M.F. (1974) Proc. Nat. Acad. Sci. USA 71, 639-643.24. Kong, P.C. and Theophanides, T. (1975) Bioinorg. Chem. 5, 51-58.25. Hermann, D. and Guschlbauer. W. (1978) Biochimie 9, 1046-1048.26. Hermann, D. (1980) Thesis, Liege, Belgium.27. Macquet, J.P. and Butour, J.L. (1978) Eur. J. Biochem. 83, 375-387.28. Nelson, J.H., Grunberger, D., Cantor, C.R. and Weinstein, I.B. (1971) J.

Mol. Biol. 62, 331-346.29. Fuchs, R.P.P. and Daune, M.P. (1972) Biochemistry 11, 2659-2666.30. Malfoy, B. and Leng, M. (1981) FEBS Letters, 132, 45-48.31. Wang, A.J.H., Quigley, G.J., Kolpak, F.J., Van der Marel, G., Van Boom,

J.H. and Rich, A. (1981) Science 211, 171-176.32. Behe, M. and Felsenfeld, G. (1981) Proc. Nat. Acad. Sci. USA 78, 1619-

1623.

5669


bernard malfoy, brighte hartmann and marc leng centre de

Documents