The site of action of 5-fluoroorotic acid on the maturation of mouse liver ribonucleic acids

Biochimica et Biophysica Actu, 319 (1973) 373-382

Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97774

T H E SITE OF A C T I O N OF 5-FLUOROOROTIC ACID ON T H E M A T U R A T I O N OF MOUSE LIVER R I B O N U C L E I C ACIDS

K. V. HADJIOLOVA, E. V. GOLOVINSKY and A. A. HADJIOLOV Institute of Biochemistry, Bulgarian Academy of Sciences, Sofia (Bulgaria)

(Received February 2nd, 1973) (Revised manuscript received April 5th, 1973)

SUMMARY The action of 5-fluoroorotic acid on the in vivo [14C]orotat e labelling of mouse liver R N A was studied. The incorporation of 5-fluoroorotic acid into polynucleotide chains has the following effects: 1. The labelling of nuclear and cytoplasmic 28-S and 18-S r R N A is almost completely inhibited, while the transcription of 45-S precursor rRNA and its early processing to 32-S and 21-S r R N A is not affected by the drug. Therefore, 5-fluoroorotic acid blocks selectively the last steps of r R N A maturation. The synthesis of nuclear 5-S r R N A is only slightly inhibited, but its transport to cytoplasmic ribosomes is blocked. 2. The labelling o f heterogeneous nuclear R N A and messenger-like RNA of cytoplasmic polysomes and free postmicrosomal ribonucleoproteins is not affected by 5-fluoroorotic acid. The drug causes the appearance of rapidly labelled degradation products in the cytoplasmic soluble RNA fraction, which indicates enhanced degradation of messenger-like RNA. 3. Administration of 5-fluoroorotic acid does not inhibit either the synthesis of nuclear precursor tRNA, its conversion into mature nuclear tRNA or the appearance o f free and ribosome-bound t R N A in the cytoplasm. It is suggested that the interference of 5-fiuoroorotic with a definite step of r R N A maturation acid may be useful in the elucidation of the molecular mechanisms of this process and of the biological effects of the drug.

INTRODUCTION The effect of 5-fluoropyrimidines on bacteria has been extensively studied, but only limited information is available about their action on animal cells (see Mandel 1). Studies with 5-fluorouracil have shown that it causes enlargement of rat liver nucleoli and a delayed release of ribosomes into the cytoplasm 2- 4. Recently, experiments Abbreviation: HnRNA, heterogeneous nuclear DNA-like RNA.

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with 5-fluoroorotic acid, a more efficient precursor of mammalian RNA, revealed 5 that the drug is extensively incorporated into messenger-like RNA of rat liver cytoplasm. Further, Wilkinson et aL 6 found that 5-fluoroorotic acid is incorporated into nuclear and cytoplasmic particles containing messenger-like ribonucleoprotein, while the labelling of 28-S and 18-S r R N A is inhibited. Since, 5-fluoroorotic acid incorporation into 45-S rRNA was apparently not affected, these authors concluded that the drug interferes with rRNA maturation. In this work the effect of 5-fluoroorotic acid on [14C]orotate incorporation into mouse liver RNA species was investigated. Our results show that 5-fluoroorotic acid does not inhibit the synthesis of 45-S precursor rRNA, but blocks selectively the last steps of rRNA maturation i.e. the conversions 32-S ~ 28-S and 21-S ~ 18-S rRNA. On the other hand, 5-fluoroorotic acid inhibits neither the labelling of heterogeneous nuclear DNA-like R N A (HnRNA) and cytoplasmic messenger-like RNA, nor the synthesis of precursor tRNA and its conversion into tRNA. Some of these results have been reported previously 7. METHODS AND MATERIALS

Isotopic labellin# Experiments were carried out with male albino mice weighing about 20 g, maintained on a standard laboratory diet. Labelling of RNA was achieved by intraperitoneal injection of 25 #Ci of [6-14C]orotic acid (spec. act. 8.5 or 16.9 Ci/mole). 30 min earlier the animals were given an equimolar dose of 5-fluoroorotic acid (3 or 1.5 #moles per mouse). In most experiments the labelling time was 3 h. Mice were sacrificed by cervical dislocation, and livers were immediately removed and placed in cold homogenizing medium. Preparation of nuclei Purified nuclei were obtained according to the procedure of Blobel and Potter a, adapted to our conditions as follows. The livers were homogenized in 2 vol. of 0.25 M sucrose in 0.05 M Tris-HCl (buffer I), pH 7.8, 0.025 M KCI, 0.01 M MgCI 2 and filtered through four layers of cheesecloth. 2 vol. of 2.3 M sucrose in buffer I were added and 28 ml of the resulting mixture were layered over 10 ml of 2.3 M sucrose in buffer I. Nuclei were sedimented at 25 000 rev./min for 45 min in the Spinco SW 27 rotor at 4 °C and the resulting nuclear pellet immediately processed further. In some experiments a crude nuclear fraction was prepared as follows. Livers were homogenized in 0.25 M sucrose in 0.025 M Tris-HCl, pH 7.4, 0.05 M KC1, 0.005 M MgCI 2. The 20 ~ homogenate was filtered through cheesecloth and spun down for 5 min at 2500 rev./min at 4 °C. Isolation and fractionation of nuclear RNA Three fractions corresponding to nuclear sap RNA, nucleolar RNA and H n R N A were extracted from purified or crude nuclei by treatment with phenol at different temperatures 9,1°. The nuclear pellet was suspended in 0.14 M NaCI and an equal volume of cold phenol saturated with 0.14 M NaCI (pH 6.0), containing 0.1 ~ 8-hydroxyquinoline, was added. The mixture was shaken for 15 min at 4 °C

5-FLUOROOROTIC ACID AND RNA MATURATION

375

and centrifuged at 5000 rev./min in the cold. RNA extracted into the water layer was further deproteinized 3-fold with cold phenol, precipitated with 96 ~ ethanol - 1 ~o potassium acetate and designated further as nuclear 4 ° RNA. The interphase layer was suspended in 0.14 M NaCI and an equal volume of phenol was added. The mixture was shaken for 15 min at 45 °C, chilled and centrifuged as above. The RNA extracted into the water phase was deproteinized 3-fold, precipitated with ethanolpotassium acetate and designated as nuclear 45 ° RNA. The interphase layer was suspended in 0.14 M NaC1 and extracted with phenol for 15 rain at 85 °C in the presence of 0.5 ~ sodium dodecyl sulphate. The mixture was chilled and centrifuged in the cold. RNA recovered from the water phase is designated as nuclear 85 ° RNA.

Isolation and fractionation of cytoplasmic RNA Fractionation of cytoplasmic ribonucleoproteins was carried out as described by Hadjiolov et al.11. The RNA from the fractions of microsomes, ribosomes and postmicrosomal ribonucleoproteins was extracted with phenol saturated with 0.2 M sodium acetate buffer (pH 6.0) in the presence of 0.5 ~ sodium dodecyl sulphate and 0.005 M EDTA. The soluble RNA components were isolated from the final supernatant after sedimentation of the postmicrosomal ribonucleoproteins (4 h at 105 000× #). The RNA from ribosomes was used to prepare low molecular weight RNA after precipitation of the high molecular weight components with 2 M NaC1. Gel electrophoresis of RNA fractions Agar gel electrophoresis and radioautography were carried out according to Tsanev and Staynov12 under conditions specified earlier 13. Since a simple correlation exists between the sedimentation coefficient of RNAs and their mobility in agar gel14, the separate components are designated further in the text by their S values. The low molecular weight RNA was analyzed also by polyacrylamide gel electrophoresis 16. Horizontal gel plates (18 cm× 10 cm) of 10 ~o acrylamide crosslinked with bisacrylamide were used. The buffer contained 7.5 mM Tris-HC1 (pH 7.8), 6 mM NaH2PO4 and 0.2 mM EDTA. The gel was prerun for 60 min, and fractionation of RNA (about 300 pg per start) was carried out at 80-100 V (about 30 mA) for 6 h at 15 °C. The gel plates were stained overnight with "all stain" (20 mg of "all stain" dissolved in 80 ml dimethylformamide and diluted to 1 1 with water)and the excess dye removed with distilled water. The gels were cut into 2-ram slices, put into counting vials and the dye bleached by exposure to direct light. The gel slices were incubated for 1 h at 60 °C in a mixture of toluene-water-Protosol (10 : 1 : 9, by vol.). After cooling, a scintillation counting medium (toluene-PPO--dimethylPOPOP) was added and the samples counted in a Packard Tri-Carb scintillation spectrometer. Reagents Analytical grade reagents were used throughout. 5-Fluoroorotic acid was synthesized in this laboratory by Mr G. Karamanov according to Chaudhuri et aL 16. Protosol was obtained from New England Nuclear; PPO, dimethylPOPOP, acrylamide and bisacrylamide from Koch-Light; sodium deoxycholate and Brij 35 from Sigma Chem. Co. The dye "all stain" was a product of Eastman Kodak Co. [6-14C]Orotic acid was obtained from the NAEC Institute for Isotopes, Budapest, Hungary.

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RESULTS A . N u c l e a r r i b o n u c l e i c acids

The effect o f 5-fluoroorotic acid a d m i n i s t r a t i o n on the overall [~4C]orotate labelling o f nuclear R N A fractions is shown in Table I. A s can be seen, the labelling o f H n R N A e x t r a c t e d at 85 °C is n o t affected b y 5-fluoroorotic acid, whilst labelling o f nucleolar R N A o f the 45 ° R N A fraction is inhibited to a b o u t 55 % o f the level in c o n t r o l mice. The 4 ° R N A fraction displays the strongest i n h i b i t i o n o f labelling by 5-fluoroorotic acid t r e a t m e n t . H o w e v e r , the low m o l e c u l a r weight R N A c o m p o n e n t s in this fraction are only slightly affected ( a b o u t 80 % residual labelling), thus indicating a s t r o n g i n h i b i t i o n in the labelling o f nuclear sap heavy R N A c o m p o n e n t s . Similar results were o b t a i n e d when the effect o f 5 - f l u o r o o r o t i c acid on the labelling o f R N A fractions f r o m purified nuclei was investigated, thus ascertaining t h a t the observed effects o f the d r u g are n o t due to c o n t a m i n a t i o n b y c y t o p l a s m i c R N A c o m p o n e n t s . TABLE 1 EFFECT OF 5-FLUOROOROTIC ACID ON THE INCORPORATION OF [14C]OROTATE INTO MOUSE LIVER NUCLEAR RNA FRACTIONS 5-Fluoroorotic acid was administered at a dose of 3 #moles per mouse 30 rain before 25 #Ci per mouse of [6-14C]orotic acid. The separate nuclear RNA fractions were extracted from nuclei by phenol treatment at different temperatures and purified further as described under Methods and Materials. Twenty mice weighing 204-2 g were used in each experimental group. Nuclear R N A fraction

85 ° RNA 45 ° RNA 4 ° RNA Low molecular weight 4 ° RNA*

Expt (No.)

1 2 I 2 1 2 1

Specific activity in cpm per A26o n,, unit Controls

÷ 5.Fluoroorotate

20 060 25 600 5 740 3 580 1 400 890 2 220

18 860 24 800 3 360 1 900 380 230 1 720

Percent inhibition

6 3 42 47 73 74 23

* This fraction was obtained from total 4 ° RNA by precipitation of the high molecular weight RNA components with 2 M NaC1 at -- 10 o and recovery of the low molecular weight RNA components from the supernatant. H e t e r o g e n e o u s nuclear R N A a n a l y z e d by a g a r gel electrophoresis reveals t h a t the labelled R N A c o m p o n e n t s in this fraction migrate m o r e slowly t h a n 28-S R N A . N o changes in the p a t t e r n o f either t o t a l o r labelled H n R N A c o u l d be detected in 5f l u o r o o r o t i c a c i d - t r e a t e d mice. N u c l e o l a r R N A e x t r a c t e d f r o m crude nuclei at 45 °C c o n t a i n s the b u l k o f the nuclear 28-S a n d 18-S r R N A c o m p o n e n t s . R a d i o a u t o g r a p h y o f the a g a r gel electrop h o r e g r a m s reveals in this R N A fraction several discrete, r a p i d l y labelled c o m p o n e n t s which m i g r a t e with mobilities c o r r e s p o n d i n g to 45-S, 32-S, 28-S, 21-S, 18-S a n d t5-S R N A (Fig. 1). I n a d d i t i o n , faint r a d i o a c t i v i t y b a n d s c a n ,be o b s e r v e d with mobilities c o r r e s p o n d i n g to 38-S, 26-S a n d 4--9-S R N A . T r e a t m e n t o f mice with 5-fluoroorotic

5-FLUOROOROTIC ACID AND RNA MATURATION

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Fig. 1. Radioautography of the agar gel electrophoregrams of mouse liver nuclear 45° RNA fraction extracted from nuclei. The separate labelled RNA components of nucleolar RNA are designated by their S valuet4 with 28-S and 18-S rRNA as standards. A, RNA from control mice; B, RNA from 5-fluoroorotic acid-treated mice. Treatment with 5-fluoroorotic acid and [14C]orotat¢ labelling as in Table I. The photoprints are overexposed to show the minor RNA components more deafly. acid abolishes almost completely the labelling of 28-S and 18-S rRNA as well as of 15-S RNA. On the other hand, the labelling of 45-S, 32-S and 21-S precursor rRNA is apparently not affected by the drug. In addition, the 26-S RNA fraction is more prominent in 5-fluoroorotic acid-treated mice. The quantitative evaluation of the changes in the components of the nucleolar 45 ° R N A fraction induced by 5-fluoroorotic acid is given in Fig. 2. As can be seen, 5-fluoroorotic acid completely inhibits the labelling o f 28-S and 18-S rRNA in parallel with 15-S RNA. On the contrary, the labelling o f 45-S, 32-S and 21-S precursor r R N A and the accompanying 26-S R N A is even higher in 5-fluoroorotic acid-treated mice, obviously due to some accumulation of these RNA components. Analysis of [~4C]orotate labelling of nucleolar RNA components obtained from purified nuclei gives essentially the same results. Nuclear low molecular weight RNA components were studied by acrylamide gel electrophoresis. The 4 ° RNA from purified nuclei contains 10 distinct low molecular weight R N A components with mobilities ranging from 4 S to 6.5 S, in good general agreement with previous findings ~9'1a. The bands of both 4-S and 5-S RNA are clearly delineated and, in addition, 4 major fractions (about 4.6, 4.8, 5.3 and 5.6 S) are observed. The labelling of the separate low molecular weight R N A fractions from control and 5-fluoroorotic acid-treated mice is given in Fig. 3. Most of the nuclear low molecular weight R N A components display low labelling a s compared with that of 5-S RNA. The only exception is the 4.6-S R N A in which the labelling is greater. The specific activities of 4.6- and 5-S RNA are 5-10-fold greater than that of 4-S RNA. Administration of 5-fluoroorotic acid does not inhibit the labelling o f either 4.6- or 4-S RNA, while that of 5-S R N A is reduced by about 50 ~ . Since it is most likely that 4.6-S R N A represents the precursor tRNA described by others ~9,2 o, the above findings indicate that 5-fluoroorotic acid inhibits neither the synthesis of precursor tRNA,

378

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nor its conversion into mature tRNA. The low labelling of the other low molecular weight RNA components does not allow any definite conclusion to be drawn about their influencing by 5-fluoroorotic acid.

B. Cytoplasmic ribonucleic acids The RNA extracted from the fractions of cytoplasmic microsomes, ribosomes and postmicrosomal ribonucleoproteins were studied. The labelling of RNA in all three fractions is inhibited by 5-fluoroorotic acid. The strongest inhibition is found in the ribosome fraction, where the labelling of RNA from 5-fluoroorotic acid-treated mice is only about 20 ~ of the level in controls. The labelling of RNA from microsomes and postmicrosomal ribonucleoproteins is reduced by 5-fluoroorotic acid to about 40-50 ~o of that in control mice. These results show that the effect of 5-fluoroorotic acid on cytoplasmic RNA labelling has a complex character. Since the RNA of purified ribosomes does not contain messenger-like RNA 1o, 1J, the above results would indicate that rRNA labelling is more strongly affected by 5-fluoroorotic acid. This assumption was confirmed by agar gel electrophoresis analysis (Fig. 4). The labelling of 28-S and 18-S rRNA in all three cytoplasmic fractions is reduced to background levels in 5-fluoroorotic acid-treated mice. On the other hand, the labelling of messenger-like RNA in the 6-S to 20-S zone remains unaltered. Thus, the results of these experiments reveal that the labelling of all cytoplasmic 28-S and 18-S rRNA components is blocked by 5-fluoroorotic acid administration, while the labelling of

5 - F L U O R O O R O T I C ACID A N D R N A M A T U R A T I O N

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Fig. 3. Chromoscan and radioactivity of low molecular weight R N A components of the nuclear 4 ° R N A fraction extracted from purified nuclei of control (A) and 5-fluoroorotic acid-treated (B) mice. Electrophoresis in I0 % acrylamide gel. The dotted line represents the radioactivity of the separate gel slices counted with the aid of Protosol in a liquid scintillation spectrometer as described under Methods and Materials. 5-ftuoroorotic acid (3/~moles/ mouse) was given 30 rain before [1,C]orotate (spec. act. 10.8 mCi/mmole) and the animals were sacrificed after 3-h of labelling. The following approximate S values were assigned to the separate R N A components: 1, 4.0; 2, 4.2; 3, 4.6; 4, 4.8; 5, 5.0; 6, 5.2; 7,5.6; 8, 6.2; 9, 6.3; 10, 6.5.

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Fig. 4. Agar gel electrophoresis and radioautography of mouse liver cytoplasmic high molecular weight R N A extracted from microsomes (A and A'), postmicrosomal ribonueleoproteins (B and B') and purified ribosomes (C and C') obtained from control (A, B and C) and 5-fluorooroticacid treated (A', B' and C') animals. 5-fluoroorotic acid was given 30 rain before~[~4C]orotate and the animals sacrificed after 3 h of labelling in vivo. - - , absorbance at 260 nm; - - -, radioactivity recorded from the radioautograms at 550 nm.

380

K . V . H A D J 1 O L O V A et al.

messenger-like RNA in polysomes and free ribonucleoproteins is little affected by the drug. Agar gel electrophoresis of the low molecular weight RNA components obtained from the RNA of ribosomes reveal that 5-fluoroorotic acid also causes an almost complete inhibition of [t4C]orotate incorporation into 5-S rRNA (Fig. 5). On the contrary, the labelling of tRNA from ribosomes is only slightly inhibited by the drug. .0.8

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Fig. 5. Agar gel electrophoresis and radioautography of mouse liver cytoplasmic low molecular weight R N A . 5-Fluoroorotic acid (6/~moles per mouse) was given 30 rain before 5 0 p C i / m o u s e (6 #moles) of [~+C]orotate. Labelling time 2 h. The low molecular weight R N A fraction f r o m ribosomes of control (A) and 5-fluoroorotic acid-treated (B) mice was obtained f r o m total R N A of ribosomes by 2 M N a C I treatment as described in Methods and Materials. The 4-S R N A fraction from control (C) and 5-fluoroorotic acid-treated (D) mice was obtained by cold phenol extraction of the final supernatant after sedimentation of postmicrosomal ribonucleoproteins at 105 000 × g for 4 h, A g a r gel dectrophoresis as given in the text, but in 2.5 % agar gel plates. - - . , absorbance at 260 nm; - - -, radioactivity recorded from the radioautograms at 550 nm,

The resistance of tRNA labelling to 5-fluoroorotic acid is also seen when the soluble RNA fraction is studied. As shown in Fig. 5 this fraction consists solely of 4-S RNA, and its labelling in 5-fluoroorotic acid-treated mice is only about 15 % lower than in controls. It should be noted that, in 5-fluoroorotic acid-treated mice, a subtantial amount of radioactive material is localized in the zone between 7-S and 18-S RNA. Since the labelling of cytoplasmic rRNA is blocked by 5-fluorooroti¢ acid, the presence of labelled material in the soluble cytoplasmic fraction is obviously due to messenger-like RNA degradation products.

5-FLUOROOROT1C ACID AND RNA MATURATION

381

DISCUSSION Previous studies s'6 have shown that labelled 5-fluoroorotic acid is extensively incorporated into rat liver RNA species. Therefore, the results obtained in the present work may be safely considered to be a consequence of 5-fluoroorotic acid incorporation into polyribonucleotide chains. In agreement with previous authors 4-6, our results show that 5-fluoroorotic acid does not inhibit the synthesis of HnRNA and 45-S precursor rRNA. In addition, we have shown that the drug also does not inhibit the synthesis of precursor tRNA. Thus it may be stated that 5-fluoroorotic acid does not interfere with the synthesis of the three main transcription products in liver cells. When further processing of these three RNA species is followed, our results clearly show that the maturation of rRNA is selectively inhibited by 5-fluoroorotic acid. Moreover, the early steps of rRNA maturation leading to the formation of 32-S and 21-S precursor rRNA are not affected by the drug, while the last steps occurring in nuclei, namely the conversions of 32-S to 28-S rRNA and of 21-S to 18-S rRNA, are almost completely blocked. Thus, the site of action of 5-fluoroorotic acid is restricted to a definite phase of the process of rRNA maturation. At present it is difficult to envisage a mechanism for the observed selective action of 5-fluoroorotic acid. It is conceivable that 5-fluoroorotic acid incorporation into RNA chains prevents some modification of the molecule which is critical for the removal of the extra pieces of 32-S and 21-S precursor rRNA by specific endo- or exonucleases. In this respect, the role of secondary methylation of some bases in the polynucleotide chain found in HeLa cells 21 and yeasts22 may be envisaged. Inhibition of 45-S and 32-S rRNA processing by methionine deprivation of HeLa cells2a further supports this assumption. Alternatively, a stringent requirement for the structure of the polynucleotide chain at the site of nuclease attack may be also considered. In any case 5-fluoroorotic acid is likely to be an important tool in studying the mechanism of the last steps of precursor rRNA maturation. The observed selective action of 5-fluoroorotic acid is the more striking if one considers our observation that the drug does not affect precursor tRNA maturation, besides the fact that the extra chain here is extremely rich in uridine residues 2°. The observed selective action of 5-fluoroorotic acid on rRNA maturation may be also important in understanding the mechanism by which the drug inhibits formation of ribosomes in bacteria ~, since in these organisms definite rRNA maturation steps have been recently uncovered. Other facets of rRNA maturation are also worth considering. The fact that the nucleolar 15-S RNA is inhibited by 5-fluoroorotic acid in parallel with 28-S and 18-S rRNA raises the question of its significance. This 15-S RNA component may be some non-conserved fragment of precursor rRNA, although the existence of such fragments has not been evidenced by previous authors 24. Further, the fact that 5-fluoroorotic acid inhibits slightly the synthesis of 5-S rRNA, but blocks its transport into the cytoplasm, indicates that 5-S rRNA cannot leave the nucleus without being integrated into the large ribosomal subparticle. Finally, it should be pointed out that although 5-fluoroorotic acid does not inhibit the synthesis and processing of messenger RNA and tRNA, the functions of these RNA species may be altered. In particular, the observation of rapidly labelled RNA components in the soluble fraction of 5-fluoroorotic acid-treated mice implies an enhanced degradation of cytoplasmic messenger-like RNA.

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A C K N O W L E D G MENTS

The authors are greatly indebted to Mr G. Karamanov for the synthesis of 5-fluoroorotic acid and to Mrs D. Kulekova for her skillful technical assistance in some experiments. REFERENCES 1 2 3 4 5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Mandel, H. G. (1969) Progr. Mol. SubcelL Biol. 1, 82-135 Stenram, U. (1966) Z. Zellforsch. Mikrosk. Anat. 71,207-216 Willen, R. and Stenram, U. (1967) Arch. Biochem. Biophys. 119, 501-503 Willen, R. (1970) Z. Physiol. Chem. 351, 1141-1150 Cihak, A. and Pitot, H. (1970) FEBS Lett. 6, 206-208 Wilkinson, D., Cihak, A. and Pitot, H. (1971) J. Biol. Chem. 246, 6418-6427 Hadjiolova, K. (1972) Studia Biophys. 31/32, 465-468 Blobel, G. and Potter, V. R. (1966) Science 154, 1662-1665 Georgiev, G. P. (1967) Prog. Nucl. Acid Res. Mol. Biol. 6, 259-351 Mackedonski, V. V., Nikolaev, N., Sebcsta, K. and Hadjiolov, A. A. (1972) Biochim. Biophys. Acta 272, 56-66 Hadjiolov, A. A., Nikolaev, N. and Shulga, A. (1972) Int. J. Biochem. 3, 509-517 Tsanev, R. and Staynov, D. Z. (1964) Biokhimiya 27, 1126-1131 Mackedonski, V. V. and Hadjiolov, A. A. (1970) Biochim. Biophys. Acta 204, 462-469 Hadjiolov, A. A., Venkov, P. V. and Tsanev, R. (1966) Anal. Biochem. 17, 263-267 Loening, U. E. (1967) Biochem. J. 102, 251-257 Chaudhuri, N. K., Montag, B. J. and Heid¢lberger, C. (1958) Cancer Res. 18,318-328 Moriyama, Y., Hodnett, J. L., Prestayko, A. W. and Busch, H. (1969) J. Mol. Biol. 39, 335-349 Prestayko, A. W., Tonato, M., Lewis, B. C. and Busch, H. (1971) J. Biol. Chem. 246, 182-187 Burdon, R. H. (1971)Progr. Nucleic Acid Res. Mol. Biol. 11, 33-79 Choe, B. K. and Taylor, M. W. (1972) Biochim. Biophys. Acta 272, 275-287 Zimmerman, E. F. (1968) Biochemistry 7, 3156-3164 Retel, J., Van Den Bos, R. C. and Planta, R. J. (1969) Biochim. Biophys. Acta 195, 370-380 Vaughan, M. H., Soeiro, R., Warner, J. R. and Darnell, J. E. (1967) Proc. Natl. Acad. Sci. U.S. 58, 1527-1534 Attardi, G. and Amaldi, F. (1970) Annu. Rev. Biochem. 39, 183-226