Pulse-polarographic analysis of double-stranded RNA

ANALYTICAL

SO, 518-530 (1974)

BIOCHEMISTRY

Pulse-Polarographic

Analysis EMIL

Institute

of Double-Stranded

RNA

PALECEK

of Biophysics, Czechoslovak Academy of Sciences, Brno 612 66 (Czechoslovakia) AND

JIRf Institute

DOSKOCIL

of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Praha (Czechoslovakia) Received November

27, 1973; accepted February 6, 1974

The reducibility of double-stranded (ds) RNA of phage f2 and its thermally denatured form was studied by the derivative (differential) pulse polarography. It was shown that the denatured RNA produced at neutral and acidic pH values a peak at negative potentials. A peak of dsRNA was much smaller and appeared at more positive potentials. The polarographic behaviour of dsRNA basically agreed with that of DNA; the peaks of dsRNA and its denatured form were however better separated. The lowest amount of the denatured RNA necessary for the determination was about 50 ng. A good sensitivity of pulse polarography for changes in RNA conformation, including damages of the RNA double helix caused by initial attack of an enzyme, low doses of ionizing radiation, etc., was demonstrated. The method was used for testing dsRNA samples prepared for biological experiments; a correlation between the polarographic behaviour of the RNA samples and their antiviral and interferon-inducing activities was found.

Synthetic and natural double-stranded polynucleotides have been rccently intensively studied for their antiviral and interferon-inducing activities (e.g., Ref. 1). During the replication of viral RNA double-stranded structure is formed (replicative form) consisting of a viral and complementary strand (2). Recently a method of isolation of double-stranded RNA1 (dsRNA) was described based on the accumulation of large amounts of dsRNA in nonpermissive cells of E. coli infected with phage f2 sus 11 (3,4). With the aid of this method it is possible to prepare purified dsRNA in large quantities. ’ Abbreviations used: dsRNA, double-stranded RNA; SSC, 0.015 M NaCl, 0.015 M sodium citrate (pH 7.0) ; 0.1 X SSC, tenfold diluted SSC; E,, summit potential of the derivative pulse-polarographic peak. 518 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

POLa4ROGRAPHY

OF

DOUBLE-STRANDED

RN.4

519

In this paper we have analyzed the abovementioned dsRNA by means of polarographic method which proved earlier as useful in the study of DNA and various biosynthetic polynucleotides (5-9). It follows from the present study that the derivat’ive pulse polarography very sensitively reflects even small imperfections in the RNA double helix and might therefore become a very important tool for the characterization of dsRNA samples prepared for biological experiments. MATERIALS

AND

METHODS

The methods of cultivation of the strain K 38 of E. coli and infection with phage f2 sus 11 were described earlier (3). dsRNA was isolated from infected bacteria 3 hr after infection either according to Billeter and Weismann (10) or Franklin (11). By its properties isolated dsRNA resembled the dsRNA samples obtained earlier from bacteria infected with wildtype phage MS2 (12). Thermal optical density profile of the sample of phage f2 dsRNA in 0.1 X SSC’ showed no changes in absorbance in the temperature range between 25-72°C. At higher temperatures a sharp transition occurred (T, = 86”C, hyperchromicity 24.9%). Sedimentation coefficient was 8.1 S and the base content 25.5 guanine, 25.5 adenine, 25.0 cytosine, and 24.0 uracil as described elsewhere (3). Acrylamide gel electrophoresis (13) showed heterogeneity in molecular weights; the samples produced several zones, corresponding to the molecular weights of lo6 to lo* daltons (14) ; molecular weight of the main part of the material was about 4.105 which corresponded roughly to the molecular weight calculated from the sedimentation coefficient 8.1. Fractions with the molecular weights lower than lo5 daltons were separated on G200 Sephadex column. Besides the dsRNA samples used for biological experiments (see Discussion) only preparations isolated according to Billeter and Weissmann (10) were used for pulse-polarographic measurements in this paper. Denatured RNA was prepared by heating of RNA solution in 0.01 X SSC, 6 min at 100°C. Precipitation of acid-insoluble material was performed with 9% perchloric acid (14). dsRNA was X-irradiated in solution in the presence of atmospheric oxygen in a TuR T250 apparatus (748 rads/min, 200 kV, 20 mA, filtration 0.5 mm of Cu and 0.5 mm of Al). Calf-thymus DNA was isolated and characterized as described previously (15). Five t.imes crystalized pancreatic ribonuclease (RNase), was purchased from Calbiochem. Polarographic measurements. The use of polarographic methods for nucleic acid studies is based on the fact that bases adenine and cytosine contained in single-stranded polynucleotides are reducible at the mercury electrode (5,6). In double-helical DNA-like polynucleotides the reducibility of these bases is suppressed. Pulse-polarographic measurements

520

PALEEEK

AND

DOSKOEIL

were performed either on A 3100 Southern-Harwell Pulse Polarograph (further A 3100) (Southern Analytical Ltd.) or on the Polarographic Analyzer PAR 174 (further PAR 174) (Princeton Applied Research Corp.) ; the latter instrument was used also for de polarography. The measurements were performed with the mercury dropping electrode in an argon or nitrogen atmosphere; as a reference electrode served a pool of mercury at the bottom of the polarographic vessel. If not otherwise stated the measurements were carried out under the following conditions: temp 27”C, drop time 2 see (auto trig) ; pulse amplitude (derivation) 50 mV; integrator signal gate 20 msec, integrator discharge 20 msec (A 3100 only). Measurements were performed in the background electrolytes containing buffered ammonium formate (5-7). To make possible a comparison between the measurements of peak heights expressed in microamperes (PAR 174) and divisions (A 3100) we measured a solution of 5.1W M Cd2+ on both instruments, using the same capillary and the conditions described above. Our measurements showed that 1 PA on PAR 174 corresponded roughly to 17 divisions on A 3100 at the amplifier sensitivity of l/32. Spectrophotometric measurements were carried out on the instruments VSU-2 (Zeiss, Jena) and Unicam SP 700. RESULTS RNA was measured in 0.3 M ammonium formate with 0.1 M sodium phosphate pH 7.0 which proved earlier as suitable for the analysis of DNA (5-7). Neither dsRNA nor its thermally denatured form (both at a concn of 300 ,&ml) produced in this medium a dc polarographic step even in the case when more sensitive current-sampled polarography was used. By means of normal pulse polarography it was possible to obtain a curve whose shape was similar to that of polynucleotides studied earlier (8,16). From the analytical point of view the most suitable were RNA polarograms yielded by derivative pulse-polarographic method (Fig. 1)) similarly as in the case of DNA (5-7). The summit potential (E,) of the derivative pulse-polarographic peak IIR of dsRNA was about 180 mV more positive than E, of peak II of native calf-thymus DNA (Table 1) E, of peak IIIR of thermally denatured RNA differed substantially less from E, of peak III of denatured DNA. Besides peaks IIR and IIIR both forms of RNA produced another peak (IR) in the vicinity of - 1.1 V (Fig. l), that is approximately at the same potential as peak I of DNA. This peak is not specific for single- or double-stranded forms of RNA as peaks IIR and IIIR and will not be studied in this communication. The difference in the height of peaks IIR and IIIR (Table 1) was not so marked as in peaks produced under the same conditions by native and denatured DNA. Further measurements have shown that for the analysis of denatured RNA the medium with pH by about one unit lower (at un-

POLAROGRAPHY

OF

DOUBLE-STRANDED

RNA

521

- III R

I R’

-1.5

-10 POTENTIAL

-03 ( V 1

030

FIG. 1. Derivative pulse polarograms of double-stranded and denatured a-+&RNA at a concentration of 250 ag/ml in 0.3 M ammonium formate with sodium phosphate pH 7. b-Thermally denatured RNA at a concentration pg/ml in 0.3 M ammonium formate with 0.1 M sodium phosphate pH 7. A amplifier sensitivity l/8.

RNA. 0.1 M of 50 3100.

changed NH,+ concn) is more convenient. Peak IIIR was more convenient. Peak IIIR was better developed2 at this pH and its E, differed sufficiently from E, of peak IIR (Fig. 2) ; thus it was possible to estimate denatured and dsRNA in the mixture. Similarly as with DNA (6)) traces of single-stranded material (less than 1%) can be estimated in dsRNA samples. For the study of peak IIR the original medium with pH 7 seems to be more suitable. In this medium the shape of peak IIR was less influenced by the presence of peak IIIR, and the height of peak IIR was practically independent of small changes in pH. At neutral pH the height ‘The increase of the height of peak IIIR with the decreasing pH is connected with the fact that the S-shaped dependence of the peak heights on pH is shifted, as compared with denatured DNA (7), to lower pH values (PaleEek, E., unpublished).

522

PALE&K

Heights, Potentials,

AND

DOSKOhL

TABLE 1 and Half-Widths of Derivate Pulse-Polarographic of RNA and DNA0

Sample dsRNA Thermally denatured RNA Native DNA Thermally denatured DNA

Concentration (rglml) 400 80 400 80

Peak IIR IIIR II III

Height 6.4

09

0.62 0.64 0.05 1.42-

-1.30 -1.48 -1.48 1.55

E,

Peaks Half-width (mV) 55 75 85

o The measurements were performed in 0.3 M ammonium formate with 0.1 M sodium phosphate pH 7 with the Polarographic analyzer PAR 174 at pulse amplitude of 50 mV. The potentials were measured against mercury pool at the bottom of the polarographic vessel.

of the peak IIIR increased with ammonium formate concentration (Fig. 3) ; a well-developed peak appeared in 0.5 M ammonium formate. The dependence of peaks IIR and IIIR in 0.3 M ammonium formate with 0.1 M sodium phosphate pH 7 on the pulse amplitude is given in Fig. 4. While in the dependence of peak IIIR a decline from linearity was observed from the amplitude of 25 mV (around pH 6 this decline was even more marked) the dependence of peak IIR was up to 50 mV almost linear. Peak IIIR (amplitude 50 mV) depended linearly on the concentration of thermally denatured RNA (Fig. 5a). At pH 5.6 and optimum setting of the pulse polarograph (high pulse amplitude, slow potential scan rate) the lowest detectable concentration of the denatured RNA was below 0.5 pg/ml (Fig. 5b). Linear dependence of the height of peak IIR on the concentration of dsRNA was observed up to about 300 &ml; at higher values the peak height was independent of concentration.

POTENTIAL

(VI

2. Derivative pulse polarogram of a mixture of thermally denatured and dsRNAs. 160 pg of dsRNA and 5 pg of denatured RNA/ml in 0.3 M ammonium formate with 0.1 M sodium phosphate pH 6. PAR 174; potential scan rate 1 mV/sec, scan range 0.75 V; current range 2 PA/scale. FIG.

POLAROGRAPHY

OF

DOUBLE-STRANDED

CONCENTRATION

RNA

523

OF HCOONH,,lM~

FIG. 3. Dependence of the height of peak IIIR on ammonium formate concentration. Thermally denatured RNA at a concentration of 25 pg/ml in 0.1 M sodium phosphate with ammonium formate at a concentration given in the graph, pH 6.8. PAR 174.

The difference in the polarographic behavior of dsRNA and its thermally denatured form makes polarography useful in the study of RNA conformational transitions. The course of thermal denaturation of dsRNA (at a concn of 78 pg/ml) followed at room temperature (after quick cooling) by means of pulse polarography and spectrophotometry is given in Fig. 6. The increase in the height of the peak IIIR at temperatures above

PULSE

AMPLITUDE

(m V 1

4. Dependence of heights of pulse-polarographic peaks of the single- and double-stranded RNAs on the pulse amplitude. (e-0) Double-stranded RNA at a concentration of 250 pg/ml in 0.3 M ammonium formate with 0.1 M sodium phosphate pH 7; (X-X) thermally denatured RNA at a concentration of 50 ag/ml in 0.3 M ammonium formate with 0.1 M sodium phosphate pH 7. A 3100; amplifier sensitivity l/8. FIG.

524

PALEEEK

0

AND

DOSKOhL

20

40

RNA CONCENTRATION

60 I #g/ml

)

FIG. 5. Dependence of the height of the pulse-polarographic peak IIIR on the concentration of the thermally denatured RNA. Measurements were performed in 0.3 M ammonium formate (e-a), with sod’mm phosphate pH 6.8 and (O-O), with 0.1 M sodium acetate pH 5.6. a-A 3100, 15 min/V, pulse amplitude 50 mV, sensitivity l/16 or lower and number of divisions calculated for sensitivity l/16. b-A 3100, 30 min/V, pulse amplitude 100 mV, sensitivity l/S.

,

40

60 TEMPERATURE

60 (‘C

1

FIG. 6. Thermal transition of the double-stranded RNA followed by pulse polarography and spectrophotometry. Samples of dsRNA at a concentration of 78 @g/ml in 0.01 x SSC were heated at the temperatures given in the graph and quickly cooled. Spectrophotometric (258 nm) ( -1, and pulse-polarographic (A-A), (peak IIR), c@-e), (peak IIIR) measurements were carried out at a concentration of 39 pg/ml in 0.3 M ammonium formate with 0.1 M sodium phosphate pH 7 at room temperature. A 3106, amplifier sensitivity l/g.

POLAROGRAFHY

OF

DOUBLE-STRANDED

RNA

525

70°C corresponded with thermal transition detected spectrophotometrically at 260 nm. The increase in the height of the peak IIR at 65°C was small but significant; if this peak was followed in dependence on time of incubation (65”C, 0.01 X SSC) its height gradually increased. After 30 min of incubation even a small peak IIIR appeared indicating the presenceof a small amount of single-stranded RNA. These time-dependent changes might be connected with chain scissionsinduced by heating. The fact that the peak IIR did not completely disappear even after heating to 90°C suggested that some double-helical regions were still contained in RNA; these might be formed due to renaturation during cooling and after addition of the supporting electrolyte at room temperature. If only peak

z4o-\

b

i5 z 5 0

2 ” ii I Y I? a

20

/:l+----

I

#

0

25

50 TIME

75

100

(MINUTES)

FIG. 7. Time-course of renaturation of double-stranded RNA. a-dsRNA at a concentration of 9.6 pg/ml in 0.01 X SSC was denatured by heating for 6 min at, 100°C and quickly cooled. The RNA solution was then mixed 1: 1 with either 5 X SSC or 2 X SSC and incubated in an ultrathermostat. Conditions of reannealing: (e---0) at 85°C in 2.5 X SSC; (0-O) at 85°C in SSC; (X-X) at 55°C in SSC. Samples were withdrawn in time intervals given in the graph and quickly cooled. Polarographic measurements were performed at room temperature at a RNA concentration of 3.2 pg/ml in 0.3 M ammonium formate with 0.2 M sodium acetate pH 5.6; PAR 174. b-(&--O) Peak IIR, (@---0) peak IIIR. dsRNA at a concentration of 108 ag/ml in 0.01 X SSC was heated for 6 min at 100°C. Then it was placed into a thermostated polarographic vessel with the same volume of 0.6 M ammonium formate with 0.2 M sodium phosphate pH 7. preheated to 58°C. The pulse polarograms were measured at 58°C in times given in the graph. A 3100, amplifier sensitivity l/8.

526

PALEEEK

AND

DOSKOEIL

IIIR is followed measurements can be performed at much lower concentrations; this is demonstrated in a renaturation experiment (Fig. 7a) in which denatured RNA at a concentration of 4.5 t&ml was reannealed at 85 and 55°C in 2.5 X SSC and 1 X SSC and peak IIR measured at room temperature. The results obtained basically agreed with those following the resistance of 32P-labeled RNA of phage MS2 to RNase (14). The reannealing can be also performed directly in the thermostated polarographic vessel (6) and changes in heights of peaks IIR and IIIR measured (Fig. 7b). As the recording of both peaks takes 5-10 min, it is however not possible to follow the initial stage of renaturation. dsRNA is susceptible to pancreatic ribonuclease at low ionic strengths (2,14), while at higher ionic strengths (above 0.15 M Na+) , dsRNA is completely resistant against the attack of this enzyme. Hydrolysis of dsRNA (at a concn of 155 pg/ml) with pancreatic RNase (1 pg/ml) was performed in SO-fold diluted SSC at 27°C and changes in the pulse polarogram (Fig. 8) followed in dependence on time. In the first 5 min peak IIR markedly increased, after 8 min an inflexion on this peak appeared suggesting the formation of a new peak with slightly more negative E, as compared with E, of peak IIR. This inflexion increased with time while the original peak IIR decreased. The presence of the inflexion caused the increase of the peak half-width. If the 30 min digested sample, which produced a marked inflexion on the pulse-polarographic curve was left at room temperature in the supporting electrolyte, the inflexion almost dis-

160

1

10 TIME

20

30

(MIN)

Fm. 8. Initial attack of pancreatic ribonuclease on dsRNA. (@-e) Height of the pulse-polarographic peak; (0-O) peak half-width. dsRNA at a concentration of 155 rg/ml in 80-fold diluted SSC was incubated with pancreatic RNAase in a concentration of 1 ag/ml at 27°C. Samples were withdrawn in the intervals given in the graph and the enzyme digestion of dsRNA stopped by the addition of the supporting electrolyte. Pulse-polarographic measurements were carried out at a dsRNA concentration of 124 pg/ml in 0.3 M ammonium formate with 0.1 M sodium phosphate pH 6.8. A 3100, amplifier sensitivity l/S.

POLAROGRAPHY

OF

0

1

DOUBLE-STRANDED

2 DOSEt

3

RNA

4

527

10

RADS 1

Fm. 9. The influence of X-irradiation on the splitting of single-stranded fragments from the double-stranded RNA. Solutions of dsRNA at a concentration of 240 pg/ml in 0.01 SSC were X-irradiated by the doses given the graph. The solutions were then heated for 10 min at 60°C and quickly cooled in an ice-bath. Polarographic measurements were performed at a RNA concentration of 180 @g/ml in 0.3 M ammonium formate with 0.1 M sodium acetate pH 5.6 on PAR 174 at a room temperature.

appeared in about 1.5 hr. The appearance of t.he inflexion whose potential differed both from 23, of peaks IIR and IIIR might be due to the presence of single-stranded material forming a part of a double-helical molecule; however further experiments will be necessary to test this presumption. In another experiment with lower RNase concentration (0.7 pug/ml) pulse polarograms were measured simultaneously with the acid-soluble fraction. In the first two minutes peak IIR increased by 20% while practically no acid-soluble fraction appeared; after 5 min this peak increased nearly by 70% (without changes in its half-width) and only 1.7% of acidsoluble material was formed. Irradiation of polynucleotide solutions results among others in scissions of the sugar-phosphate backbone (17). If the distance between two singlestrand scissions in the double-helical polynucleotide is sufficiently small the stability of the double helix is disturbed and single-stranded fragments may be splitted off (17,18). The formation of single-stranded fragments is stimulated by elevation of temperature and by low ionic strength. dsRNA at a concentration of 240 pg/ml in 0.01 X SSC was X-irradiated, then heated at 60°C and polarographed at room temperature at pH 5.6 (Fig. 9). Peak IIIR indicating the presence of single-stranded material increased linearly with the radiation dose in the range of 5OCMOOO rads. DISCUSSION

From among double-helical poly(r1) * (rC), and poly(rG) pulse polarography (5,6,9).

polyribonucleotides so far poly (rA) - (rU) , . (rC) have been analyzed with the aid of In comparison with their parent single-

528

PALEEEK

AND

DOSKOhL

stranded polyribonucleotides containing reducible bases/poly (rA) reap. poly (rC)/double-helical polyribonucleotides produced substantially smaller pulse-polarographic peaks, Poly (rG) * (rC) yielded no peak at usual concentrations (9). Peaks of poly (rA) * (rU) and poly (r1) . (rC) appeared at more positive potentials than peaks of single-stranded poly (rA) resp. poly (rC) . In these respects pulse-polarographic behavior of dsRNA thus corresponds both with the behavior of the abovementioned biosynthetic polyribonucleotides and with that of DNA (Table I), poly (dA-dT) * (dA-dT) and poly (dG) * (dC) (9). The absence or the decrease of pulsepolarographic current as a result of double helix formation was explained by hiding of the reduction sites in the interior of the molecule (5-7). For low currents produced by double-helical polynucleotides (e.g., peak IIR of dsRNA) small amounts of bases, forming parts of “opened” regions (which differ by their conformation from the rest of the molecule) might be responsible. These regions are located at the ends of the molecule, in the vicinity of single-strand interruptions and probably in the neighborhood of other imperfections of the double helix (5-7). Bases which form part of sufficiently long sequences of rG-rC or dG-dC pairs are completely inaccessible for the electrode process (9). Our recent studies of the transition of poly (rA) into its protonated double-helical form (19) suggest that the formation of the double helix is not the sufficient condition for the inaccessibility of the reduction sites for the electrode process. In the protonated helix of poly (A) the reduction sites in adenine residues do not participate in hydrogen-bonding, they are localized close to the surface of the molecule (8) and are polarographically reducible (19). The nonlinear dependence of the height of the pulse-polarographic peak of dsRNA on the pulse amplitudes (Fig. 4) differs from dependences observed with simple inorganic substances (20) ; it is similar, however, to the dependences obtained with poly(rC) (8), poly (rA) (16)) and DNA (PaleFek, E., unpublished). It was presumed that nonlinearity of the abovementioned dependence is connected with accumulation of the polynucleotide in the electrode surface at more positive potentials than the reduction potential (8) and that the course of this dependence is influenced by the difference between the desorption and reduction potentials of the given polynucleotide. The main contribution of this paper lies in the finding that the derivative pulse polarography very sensitively reflects conformational changes of dsRNA including minor changes caused by the initial attack of an enzyme, low doses of X-radiation, heating, etc. By means of this method it is possible to study denaturation and renaturation of dsRNA (Figs. 6,7) in concentrations comparable with those used in experiments in which the resistance of 32P-labeled RNA to RNase is followed as a criterion of

POLAROGRAPHY

OF

DOUBLE-STRANDED

RNA

529

double helicity (14). At higher dsRNA concentrations the presence of few tenths of percent of the single-stranded material can be easily detected. This fact can be utilized for the detection of chain scissions in dsRNA (Fig. 9). Introduction of chain scissions is also indicated by the increase of peak IIR (Fig. 8). The estimation of single-strand breaks in DNA does not present any difficulties. Alkali-lability of RNA excludes, however, the use of alkalic gradients, therefore pulse polarography may become the only technique capable of detecting small damages of double-stranded polyribonucleotides. Pulse-polarographic measurements can be conveniently performed in few tenths of a milliliter in a simple microvessel. At the pulse ampIitude of 100 mV in a suitable medium (e.g., 0.3 M ammonium formate with sodium acetate pH 5.6) thermally denatured RNA can be detected in a concentration of about 0.2 pg/ml (Fig. 5b) ; thus the lowest quantity of RNA necessary for the analysis is about 50 ng. High sensitivity of the derivative pulse polarography for the detection of small changes in dsRNh conformation caused by various agents stimulated our work with dsRNA samples prepared for biological experiments. A comparison of pulse polarograms of these samples with their antiviral and interferon-inducing activities showed that the biological activity was dependent on the intact secondary structure of dsRNA indicated by nonenlarged peak IIR at neutral pH and by the absence of peak IIIR. It followed from the results of testing the protecting effect against the experimental infection by tick-borne encephalitis virus (21) that the most active were the samples prepared according to Billeter and Weissman (10,141 (i.e., using RNase at high ionic strength) which produced only peak IIR. Somewhat less active were samples prepared according to Franklin ( 11) which produced besides peak IIR also higher or lower peak IIIR. Besides bihelical structure another condition for antiviral activity was sufficiently high molecular weight, which should be above 1.105 daltons. Preliminary experiments have shown that the presence of the polarographically detectable single-stranded material in dsRNA preparat,ions unfavorably affects their interferon-inducing ability in tissue culture of 1, line of mouse fibroblasts (Jandejsek, ,J., unpublished). The biological activity of double-stranded polyribonucleotides has been intensively studied, however, the intactness and purity of samples have not yet been standardized. It follows from our experiences so far obtained, that for the characterization of dsRNA samples the most important is the derivative pulse polarography and the gel electrophoresis (characterizing t.he distribution of molecular weights). With the aid of the former technique it is possible, besides the facts mentioned in this paper, to detect traces of proteins (22), in several micrograms of a nucleic acid sample. Pulse polarography does not yet belong among methods commonly used

530

PALEEEK

AND

DOSKOEIL

in nucleic acid research. One of the main reasons of commercially available low-cost instruments. recently overcome (23) and one can hope that polarography for the analysis of nucleic acids will

of this state was the lack This difficulty has been the advantages of pulse be soon fully recognized.

ACKNOWLEDGMENT The authors wish to thank Mrs. I. Postbieglova

for her skillful

technical assistance.

REFERENCES A. K., LAMPSON, G. P., TYTELL, A. E., NEMES, M. M., AND HILLEMAN, M. R. (1967) Proc. Nut. Acad. Sci. US 58, 2102-2108. 2. WEISSMAN, C., AND OCHOA, S. (1967) in Progress in Nucleic Acid Research and Molecular Biology (Davidson, J. N. and Cohn, W. E., eds.), Vol. 6, pp. 353-399, Academic Press, New York. 3. DOSKOEIL, J., FUCHSBERGER, N., VETR~K, J., LACKOVIE, V., AND BORECKJ, L. (1971) 1. FIELD,

Acta Viral. 15, 523. 4. FUCHSBERCER, N., VETRAK, J., LACKOVIE, V., BORECK?, L., AND DOSKOEIL, Acta Viral. 16, 466476. 5. PALE&K, E. (1969) in Progress in Nucleic Acid Research and Molecular

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Biology (Davidson, J. N. and Cohn, W. E., eds.), Vol. 9, pp. 31-73, Academic Press, New York. 6. PALEEEK, E. (1971) Z~L Methods in Enzymology (Grossman, L. and Moldave, K., eds.), Vol. 21, pp. 3-24, Academic Press, New York. 7. PALEEEK, E., AND BRABEC, V. (1972) Biochim. Biophgs. Acta 262, 125-134. 8. PALEEEK, E. (1972) Coil. Czech. Chem. Commun. 37, 3198-3208. 9. BEZD&KOV~~, A., AND PALEEEK, E. (1972) 10. BILLETER, M. A., AND WEISSMAN, C.

search (Cantoni, New York. 11. FRANKLIN,

Stud&z

Biophys.

34,

141-149.

(1966) in Procedures in Nucleic Acid ReG. L. and Davies, D. R., eds.), pp. 498-512, Harper and Row,

R. M. (1966) Proc. Nat. Acad. Sci. US 55, 1504-1511. 12. DOSKOEIL, J., AND PALEEEK, E., in preparation. 13. PEACOCK, A. C., AND DINCMAN, C. W. (1968) Biochemktr~ 7, 668-674. 14. BILLETER, M. A., WEISSMAN, C., AND WARNER, R. C. (1966) J. Mol. Biol. 17, 145-173. 15. BRABEC, V., AND PALEEEK, E. (1970) Biophysik 6,290-300. 16. BRABEC, V., AND PALEEEK, E. (1973) 2. Naturforsch 28c, 685-692. 17. Lu~Ai3ov& E., AND PALEEEK, E. (1971) Rad. Res. 47,51-65. 18. Lutiijov~, E., AND PALEEEK, E. (1972) Foliu Biol. 18, 307-316. 19. PALE~EK, E., VETTERL, V., AND SWNAR, J. (1974) Nucleic Acid Res. 1, 427442. 20. PARRY, E. P., AND OSTERYOUNG, R. A. (1965) Anal. Chem. 37, 1634-1637. 21. GAJDOF~OVL, E., DOSKO~IL, J., AND MAYER, V. (1973) Acta Viral. 17, 196292. 22. VORL~~KOV& M., AND PALE&K, E. (1973) Biochim. Biophys. Acta 331, 27&282. 23. FLATS, J. B. (1972) Anal. Chem. 44,75A-87A.