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On the structural alterations of phage proteins synthesized in the presence of pancreatic ribonuclease
VIROLOGY
19,
169-178 (1963)
On the Structural in the
Alterations Presence
of Pancreatic
R. JEENER Laboratory
of Animul
of Phage
Synthesized
Ribonuclease
G. VANSANTEN
AND
Physiology, Accepted
Proteins
University
October
of Brussels,
Belgium
5, 1962
The addition of pancreatic ribonuclease to a lysogenic culture of Bacillus megaterium, induced by ultraviolet irradiation, leads to the formation of noninfectious phage particles of normal deoxyribonucleic acid content. The stability of the tail structure is sharply decreased. The head proteins, dispersed by a sonic oscillator, fail to react with the specific antibodies of control phage. The peptides obtained by tryptic digestion give a distinctly abnormal “fingerprint” by chromatography and electrophoresis. The hypothesis is put forward that the ribonuclease acts by modifying the specificity of the ribonucleic acids involved in the ordering of amino acids along the genetic messenger. INTRODUCTION
As we have briefly reported (Jeener et al., 1960)) the addition of bovine pancreatic ribonuclease (RNase) to an induced lysogenie culture of Bacillus megaterium leads to the production of noninfectious phage, the various proteins of which have lost their usual immunological properties. Here we present the results in detail and show that t’he proteins synthesized in the presence of RNase seem to have a different primary structure. The modifications of protein structure studied do not occur if the enzymatic active center of the RNase is blocked. It seems probable, therefore, that the RNase is acting as an enzyme rather than simply as a basic protein. The most likely hypothesis is that it brings about an alteration in one of the fractions of ribonucleic acid (RNA) involved in establishing protein specificity. One would expect the effect of RNase on a genetic messenger to be solely destruction or inactivation, causing a decrease in the rate of protein synthesis, but not an appearance of abnormal proteins. If, however, RNase acts on the amino acid transfer RNA by modifying the specificity of its constituents, the resulting alterations of the decod-
ing system could lead to the appearance of abnormal proteins by introducing systematic errors in the ordering of the amino acids, the information carried by the deoxyribonucleic acid (DNA) remaining unaltered. It may be noted that analogous results have been obtained by the action of purine or pyrimidine analogs in other systems (Chantrenne, 1958 ; Chantrenne and Devreux, 1960; Hamers and Hamers-Casterman, 1961; Naono and Gros, 1960a,b; Bussard et al., 1960; Benzer and Champe, 1961), MATERIAL
AND METHODS
The lysogenic and sensitive strains of Bacillus megaterium used, the culture and induction techniques, and the method of phage titration have been described previously (Jeener, 1958a,b). The bovine pancreatic RNase (Sigma) is added to the cultures 20 minutes after induction, that is, at, the time when synthesis of phage protein begins. If it is added earlier, there is too great an inhibition of phage protein synthesis (Jeener, 195813)) preventing the harvesting of adequate amounts of material. The concentration of RNase used is chosen (unless otherwise specified) such
169
170
JEENER
AND VANSANTEN
that lysis is retarded by at least 2 hours and that the number of plaques is reduced 30300 times per unit weight of phage DNA. The latter is measured by analysis of the immune precipitate from a known volume of lysate. The amount of RNase required varies both with the RNase preparation and with bacterial strain; it must be determined before each series of experiments. It may be mentioned here that RNase added to an induced culture which has already lysed does not produce any of the effects to be described below. Thus RNase is totally without direct effect on complete phage particles. Ss5-labeled phage are prepared by the addition of S35-methionine (Radiochem. Centre, Amersham, England) immediately after induction of a culture, in the usual medium from which casein hydrolyzate has been omitted. The starting material for the preparation uf purified phage is a partially or totally lysed culture to which have been added lysozyme (1 mg per 100 ml) and chloramphenicol. This lysed culture is subjected to three cycles of centrifugation, alternately at 3500 g and at 65,000 g. The cfficiency of purification is controlled by electron microscopy, which shows only t’ract impurities at magnification ranging to 40,000 X. In some cases, the presence of contamination with bacterial constituents was estimated, by purification of unlabeled phage in the presence of a lysozyme lysate of sensitive S3s-labeled bacteria, to be at most 2%. Estimation of phage DNA was carried out by the method of Ceriotti (1952). To compare the ability of different phage preparations t,o adsorb onto sensitive bacteria, S3”-labeled phage was added to a growing culture in the proportion of one phage to 10 bacteria. At intervals between 0 and 10 minutes, the bacteria were centrifuged, and the serum-precipitable radioactivity of the supernatant was measured. Examination of phage with the elect,ron microscope (Siemens Elmiskop) was done after shadowing with palladium or using the phosphotungstic acid negative staining technique.
The immunological techniques used are described with the experimental results. When dispersed particles were used, the dispersion was done by treating the phage suspension in a thin-walled Lusteroid tube for 12 minutes in a sonic oscillator Raytheon, 250-watt, IO-kc, at maximum intensity. The duration of the sonic treatment was such that no longer were structures distinguishable under the electron microscope, nor did centrifugation at 65,000 g for 30 minutes produce any appreciable sedimentation of phage material. The technique chosen for the preparation of tryptic peptides is that used by Brenner et al. t.1959) when the amounts of phage proteins available are very small (denaturation at pH 2, treatment with deoxyribonuclease, digestion by trypsin in presence of 2% NH4HCOs, pH 8). The separation of peptides was carried out by chromatography and by electrophoresis on Whatman filter paper 3 MM under the conditions described by Katz et al. (1959) for the separation of tryptic and chymotryptic peptides of phage T2. RESULTS Comparison between Particles Cultivated in the Presence of RNase and Kormal Particles If RNase is added to induced cultures of B. megaterium lo-20 minutes after induction, that is, at the time when phage pyotein synthesis begins, there is a decrease both in the amount of phage produced and in the infectivity (plaques per unit weight of DNA) of the particles. Figures l-3 present the results of a particularly clearcut experiment. The number of plaques per unit weight of phage DNA decreases approximately linearly as the concentration of RNase increases. Growth of the bacteria after induction is normal until lysis, which is complete although somewhat delayed. With concentrations of RNase of 50 to 100 pg/ml, lysis becomes incomplete, the number of plaques per unit weight of phagc DNA being reduced by as much as 300 times (see below). In the above experiment the quantity of
ALTERED
PHAGE
PROTEKS
phage was measured by the amount of DNA precipitated by addition of antiserum to a known volume of lysate. In the experiments that follow, the phage particles were purified by three successive cycles of centrifugation. These preparations examined by electron microscopy contained no appreciable amounts of bacterial debris. They were immediately examined for numbers of plaques per unit weight of DNA, for the proportion of particles adsorbed onto sensitive bacteria, and for capacity to fix neutralizing antibody. The results of these tests may be summarized as follows: On the one hand, there is a reasonable parallelism between the number of plaques per unit weight of DNA and the number of particles adsorbed. For example, in three experiments in which infectivity decreased 5-, 21- and 253-fold, respectively, the proportion of adsorbed particles diminished, respectively, from 86% to
1
/
I
I
UNDER
RNASE
RIBONUCLEASE
AODED
lpglml
I
FIG. 2. Quantity of phage (dry weight of precipitate obtained with antiphage serum) and number of plaque-forming particles produced per milliliter of cultures in the presence of various concentrations of RNase (same experiment as Fig. 1). Quantity of phage and number of infectious particles are expressed as percentages of the values for a control culture without RNase.
1 ,
I .
171
ACTION
I
I
O/bg RNaso
0 10/q
”
r g ”” 030/q A20
I 0
I 10 RIBONUCLEASE
I
20 ADDED
‘jtg
I JO I ml 1
J LO
FIG. 3. Infectivity of phage produced in the presence of various concentrations of RNase (same experiment as Figs. 1 and 2). Infectiv-ity is measured by the number of plaques corresponding to the same weight of DNA precipitable by antiphage serum. b I
I
I
I
_I
FIG. 1. The optical density of lysogenic cultures induced at time zero after addition of different concentrations of RNase at the time indicated by an arrow.
20%, 90% to 5%, and 97% to nonmeasurable value. The loss of capacity to adsorb adequately explains the low infectivity of particles produced in the presence of RNase. On the other hand, there is no parallelism between the number of plaques and the capacity to fix neutralizing antibodies. As the
172
JEENER
AND
VANSANTEN
Fro. 4A FIG. 4. Preparations of phage purified by three cycles of centrifugation. cultured in the presence of RNase; C, cultured in the presence of RNase, where tails remained attached.
concentration of RNase increases, the capacity to fix neutralizing antibodies is reduced at most 3 times, whereas the number of infectious particles decreases down to l/300 the normal value. Hence, particles that are neither infectious nor able to adsorb onto sensitive bacteria are nevertheless able to fix neutralizing antibodies. Electron microscopy of particles produced in the presence of RNase usually reveals that most particles have lost the long tail that is characteristic of the phage studied (Fig. 4, A and B). We believe that the loss of the tail may result from mechanical effects during spreading and dehydration on the electron microscope grid for the following reasons. Occasionally, phage preparations whose infectivity was reduced 30 to 300 times showed a majority of particles with a tail, but the tail was pulled away from the head and attached to it by a fine filament (Fig.
A, control; 13, except)ional case
4C). In other cases, intact tails separated from the heads and clumped together were clearly recognizable; the number of tails identifiable represented 50% of the number of heads. Also, if it is assumed that the ability to fix neutralizing antibodies resides in tail constituents in the phage (as in the case for phage T2 of Escherichin coli) (Lanni and Lanni, 1953)) the purified noninfectious particles that can fix considerable amounts of neutralizing antibodies must still have a tail. Finally, the ratio of DNA to protein in purified particles was always the same in normal particles and in those cultured in the presence of RNase. Had the tails been absent, the ratio of DNA to proteins would probably have been modified significantly. Hence it may be concluded that phage cultured in the presence of RNase differ from normal phage by their inability to adsorb onto sensitive bacteria and by the lower stability of the links between head and tail.
ALTERED
PHAGE
PROTEINS
UNDER
Fm. 4B
FIG. 4C
RNASE ACTIOX
173
174
JEENER
Phage
AND VANSANTEN
proteins
added kountslmin)
FIG. 5. Antiphage serum precipitation curves for proteins of normal phage (curve 1) antI of phage grown in the presence of RNase concentrations of 40 pg/ml (curve 2) and 60 pglml (curve 3). The protein preparations had the same specific radioactivity (S”). The proteins added and precipitated were measured by their radioactivity.
Immunological Properties Compared An immunological examination of phage constituents other than those which fix neutraliaing antibody was done after sonic disruption of the particles. Phage were cultured in the presence of Ss5-methionine added at the time of induction. The phage particles were purified, sonicated, and added in increasing amounts to a constant quantity of a rabbit antiserum against purified normal phage (diluted &j-fold with 0.9% NaCl). After 2 hours at 37” and 48 hours at 4”, the precipitates were centrifuged and washed with 0.9% NaCl. The radioactivity was measured in the precipitates and in the supernatants. With disrupted control phage, maximum precipitation completely exhausted the antibody, as shown by tests on the supernatant. Hence, the sonic treatment does not destroy sponding antibodies. With proteins derived from phage cultured in the presence of increasing concentraticns of RNase, there was a progressive decrease in the maximum amount of precipitation, as shown in Fig. 5. These results indicated that the proteins of the head were quite definitely altered, like those of the tail. The progressive decrease of precipitation with increasing con-
the disappearance at unequal rates of the various reactive groups carried by the different phage proteins. As a control, absorption experiments were carried out to determine the amount of antibody fixed by equal weights of normal proteins and of proteins from phage grown in the presence of RNase. Mixtures of antiserum, appropriately diluted in 9% NaCl with increasing quantities of sonically disrupted preparations were incubated for 2 hours at 48”, then left, at 4” overnight. The precipit,ates were separated by centrifugation, and the supernatant,s were collected. Each supernatant then received a constant amount of control phage protein labeled with S:j”methionine such that 95% would be precipitated by the initial unabsorbed amount of antibody. After 2 hours at, 37” and overnight in a refrigerator, the proteins were centrifuged and washed and the radioactivity was measured in the precipitate and in the supernatant. The proportion of antigen precipitated served as a measure of the amount of the antibody that remained in the supernatant after the absorption procedure. Results are presented in Figs. 6 and 7. The antibody-fixing power of the antigens can be compared by the amounts that are needed to leave 50% of -the Ss5 unadsorbed. It is clear that the amount of phage
centrations
protein
the affinity
of their
of RNase
proteins
for the corrt-
could be explained
by
needed
to fix
a given
amount
of
ALTERED
PHAGE
PROTEINS
antibody increases with increasing concentrations of RNase present during protein synthesis. For the highest concentration of RNase that we could use, the proteins fix I1 0 78 E GE
B .s Ix .; 'd h & 21 =D' = E <.; E z %
10 9-
I
a: oyg
RNarelmt
o:6Opg
”
I
I,,,,. 0
6-
,b-
5432' %
I I I I 20 30 40 50 test
lo-20 times less antibody than equal weights of normal protein. These findings confirm that the phage head proteins have been changed considerably, since the number of antibody combining groups per unit weight of protein is greatly reduced. Test of RNase Inactivated Acid
7-
10 of
175
RNASE ACTION
c
a-
0
UNDER
phdge
protein
,
I
I
I 100
nonprecipitated
FIG. 6. Fixation of antibody by the proteins from control phage and from phage synthesized in the presence of RNase. First part : various amounts of sonic extracts from normal phage and from phage grown in the presence of RNase were added to a fixed amount of antibody. Second part: fixed amounts of S%-labeled test phage are added. The percentage of label not precipitated is estimated.
by Bromoacetic
It is important to test whether t.he RNase functions by virtue of its enzymatic activity or by some different property, for example, its basic character. Since carboxymethylation of one histidine residue causes complete inactivation of RNase without denaturing it (Barnard and Stein, 1959)) in order to study the effect of an inactive form of RNase, experiments were carried out using either normal enzyme or enzyme 90% inactivated by this method. Comparison of optical density after induction, of the amount of phage produced, of the number of plaques per unit weight of phage, and of the amount of antibody fixed per unit weight of phage protein showed that the inactivation of the RNase completely suppressed the effects of untreated RNase (see Fig. 7). There is little doubt therefore, that the effects of RNase described are due to its enzymatic activity. That RNase added to the culture medium
1 !,:
0 Org RNase/ml A 6Ojcg " 0100p "
, 1
,
(inactivate
q 1oo/Lg ‘.
; i
lo 20 30 40 % of tort phage protein
50 60 70 nonprecipitated
80
90
1010
FIG. 7. Effects of the inactivation of RNase by bromoacetic acid on the ability of phage proteins to fix antibody (see Fig. 6 legend for the technique used).
176
JEENER
AND
0
0
*
0
VANSANTEN
be profoundly altered. A study of the nature of localized changes in the proteins was undertaken to help clarify the mode of action of RNase. Fingerprints obtained by two-dimensional electrophoresis and chromatography of tryptic peptides of RNase-grown phages were different from those of control phages. Denaturation and trypsin digestion were performed under identical conditions and led to reproducible results. However, in view of the very small amount of protein available, it would seem hazardous at present to draw any conclusions as to the origin of the differences. These differences confirm, however, the abnormal character of the proteins synthesized in the presence of RNase. DISCUSSION
00 FIG. 8. Paper electrophoresis and chromatography of the peptides liberated by trypsin (continuous lines: the most clearly visible peptides obtained from normal phage protein. Dotted lines: the most clearly visible peptides from proteins of phage cultured in the presence of RNase). Electrophoresis under 2000 volts for 75 minutes at pH 3.7; chromatography in n-butanol-acetic acid-water (4: 1: 5).
can act enzymatically inside the cells of B. megaterium has been shown by Y. Robin (1962)) who observed that increasing concentrations of RNase cause a progressive decrease of the nonribosomal RNA up to 45%, ribosomal RNA and DNA remaining unaffected. No such effect was observed with enzyme inactivated by the method of Barnard and Stein. The Nature of the Modifications by the Phage Proteins
Undergone
The modified phage proteins can still aggregate and form particles. Their surface properties must therefore be fairly close to normal. Hence it seemed probable that the folding of the polypetide chains would not
It seems unlikely that RNase leads to the synthesis of abnormal proteins by modifying the information carried by the phage DNA. If that were the case, RNase would be a powerful mutagenic agent, a possibility not supported by examination of the plaques produced by the surviving phage part’icles. The fact that RNase inactivated by bromoacetic acid is altogether without effect favors the idea that it’ acts enzymatically either on the messenger RNA which directs the synthesis of phage proteins, or on the S-RNA which orders the amino acids on the messenger. The hypothesis that the RNase acts on the messenger RNA, causing formation of abnormal proteins, is doubtful; for it could only fragment the messenger, giving rise to pieces which would either be inactive or produce incomplete polypeptide chains. It is hard to imagine that such chains could fold into tertiary structures normal enough to aggregate and form particles of normal appearance. More tempting is the hypothesis that the RNase acts on the S-RNA. These nucleic acids are stable (Hoagland and Comly, 1960). During our experiments, they were constantly exposed to the attack of RNase whereas the messenger RNA, which probably has a rapid turnover (Jacob and Monod, 1961) would be less likely to be acted upon by the enzyme before it functioned as a messenger.
ALTERED
PHAGE
PROTEINS
The fact that RNase applied in viva under our conditions decreases by up to 45% the S-RNA precipitable by trichloroacetic acid (Robin, 1962) suggests that some of S-RNA molecules that are still precipitable by trichloroacetic acid are also somewhat damaged. If some of the S-RNA fragments that are preferentially produced by RNase (Rushieky et al., 1961) had new specificities, either placing their normal amino acids at abnormal points on the messenger or fixing the wrong amino acid to their normal position, it would be possible to explain the changes in immunological properties of the proteins synthesized in the presence of RNase. Then we would have to admit that the mechanism of decoding of genetic information can be modified by enzymatic activity, the character of the resulting proteins depending on the specificity of the enzyme. It would be interesting to carry similar experiments using ribonucleases of various specificities. N. DuPont-Mairesse in our laboratory carried out a series of experiments which indicated that a large part of the phage proteins synthesized in the presence of RNase are unable to assemble into phage particles (DuPont-Mairesse, 1962) but are recognizable by their capacity to combine with antiphage antibodies. In contrast, the proteins we studied could combine to form particles but had lost their reactivity with the antibodies. Thus, the action of RNase may lead to modification either of the immunologically reactive site or of the sites by which
the molecules
combine
to form
phage particles. Abnormal molecules carrying both defects, if present, would have escaped detection in our experiments. ACKNOWLEDGMENTS This work was made possible by the efficient technical assistance given by Mr. R. De Neyn. Dr. Beaufays and Dr. Vanderhaeghe gave indispensable help with the electron microscope studies. The interpretation of the results is the fruit of many discussions with Dr. De Deken-Grenson and Dr. R. Hamers. Our colleague H. Chantrenne kindly read the text and made many useful suggestions. The manuscript was translated by Miss Hell. To all of them, the authors express their warmest thanks.
UNDER
177
RNASE ACTION
The work was carried out under the association contract 016-61-10 ABIB between Euratom and the Universitd libre de Bruxelles. REFERENCES BARNARD, E. A., and STEIN, W. D. (1959). The
histidine residue in the active centre of ribonuclease. I. A specific reaction with bromoacetic acid. J. Mol. Biol. 1,339-349. BENZER,S., and CHAMPE,S. P. (1961). Ambivalent rI1 mutants of phage T4. Proc. N&t. Acad. Sci. U. S. 87,1025-1038.
BRENNER, G., STREISINGER,G., HORNE, R. W., CHAMPE, S. P., BARNETT, I,., BENZER, S., and REES, M. W. (1959). Structural components of bacteriophage. J. Mol. Biol. 1, 281-292. BUSSARD,A., NAONO,F., GROS, F., and MONOD,J. (1960). Effets d’un analogue de l’uracile sur les propriktes dune proteine enzymatique synthktisee en sa presence. Compt. rend. acad. xi. 250, 404s4051.
CERIOTTI, G. (1952). A microchemical determination of deoxyribonucleic acid. J. Biol. Chem. 198,
297303. CHANTRENNE, H.
(1958). La 8-azaguanine provoque-t-elle la formation de proteines anormales. Biochem. Pharmacol. 1,233-234. CHANTRENNE, H., and DEVREUX, S. (1960). Restauration de la synthese d’enzymes apres inhibition par I’azaguanine. &o&m. et Biophys. Acta 41,239-245. DUPONT-MAIRESSE, N. (1962). On the abnormal character of phage precursor proteins synthesized in presence of ribonuclease. Biochim. et Biophys. Acta 61, 129-134. HAMERS, R., and HAMERS-CASTERMAN, C. (1961). Synthesis by Escherichia coli of an abnormal p-
galactosidase in the presence of thiouracil.
J.
Mol. Biol. 3, 166174.
HOAGLAND,M. B., and COMLT, L. T. (1960). Interaction of soluble ribonucleic acid and microsomes. Proc. Natl. Acad. Sci. U. S. 46, 1554-1563. JACOB,F., and MONOD,J. (1961). Genetic regulatory mechanisms in the synt.hesis of proteins. 1. Mol. Biol. 3,318356. JEENER, R. (1958a). The action of ribonuclease on
sensitive
and non-induced
lysogenic
cells of
Bacillus megaterium. Biochim. et Biophys. Acta 32,99-105. JEENER, R. (1958b). The action of ribonuclease on
phage protein synthesis by an induced lysogenic Bacillus megaterium culture. Bioehim. et Biophys. Acta 32,106116. JEENER, It., DUPONT-MAIRESSE, N., and VANSANTEN, G. (1960). Proteines anormales de phages synthetisees en presence de ribonuclease. Biochim. et Biophys. Acta 45,245-255. KATZ, A. M., DREYER, W. J., and ANFINSEX, C. B.
178
JEENER
AND VANSANTEN
(1959). Peptides separation by two-dimensional chromatography and electrophoresis. J. Biol. Chem. 234,2897-2900. LANKI, F., and LANNI, Y. THBRY (1953). Antigenic structure of bacteriophage. Cold Spring Harbor Symposia Quant. Biol. 18,159-168. NAONO, S., and GROS, F. (1960a). Effets d’un analogue de base nuclkque sur la biosynthke de protknes bactbriennes. Changements de la composition globale. Compt. rend. acad. xi. 250, 35273529. NAOKO, S., and GROS, F. (1960b). Synthke par
Escherichia coli d’une phosphatase modifike en prksence d’un analogue pyrimidique. Compt. rend. acad. sci. 250,3889-3893. ROBIN, Y. (1962). Relation entre la tcneur des cellules de Bacillus megaterium cn acide ribonuckique non ribosomial et la I-itesse de synthke des protknes. Biochim. et Biopkys. Acta 55,556-557. RUSHIZKY, G. W., KNIGHT, C. A., and SOBER, H. A. (1961). Studies on the preferential specificity of pancreatic ribonuclease as deduced from partial digests. J. Biol. Chem. 236, 2732-2737.
19,
169-178 (1963)
On the Structural in the
Alterations Presence
of Pancreatic
R. JEENER Laboratory
of Animul
of Phage
Synthesized
Ribonuclease
G. VANSANTEN
AND
Physiology, Accepted
Proteins
University
October
of Brussels,
Belgium
5, 1962
The addition of pancreatic ribonuclease to a lysogenic culture of Bacillus megaterium, induced by ultraviolet irradiation, leads to the formation of noninfectious phage particles of normal deoxyribonucleic acid content. The stability of the tail structure is sharply decreased. The head proteins, dispersed by a sonic oscillator, fail to react with the specific antibodies of control phage. The peptides obtained by tryptic digestion give a distinctly abnormal “fingerprint” by chromatography and electrophoresis. The hypothesis is put forward that the ribonuclease acts by modifying the specificity of the ribonucleic acids involved in the ordering of amino acids along the genetic messenger. INTRODUCTION
As we have briefly reported (Jeener et al., 1960)) the addition of bovine pancreatic ribonuclease (RNase) to an induced lysogenie culture of Bacillus megaterium leads to the production of noninfectious phage, the various proteins of which have lost their usual immunological properties. Here we present the results in detail and show that t’he proteins synthesized in the presence of RNase seem to have a different primary structure. The modifications of protein structure studied do not occur if the enzymatic active center of the RNase is blocked. It seems probable, therefore, that the RNase is acting as an enzyme rather than simply as a basic protein. The most likely hypothesis is that it brings about an alteration in one of the fractions of ribonucleic acid (RNA) involved in establishing protein specificity. One would expect the effect of RNase on a genetic messenger to be solely destruction or inactivation, causing a decrease in the rate of protein synthesis, but not an appearance of abnormal proteins. If, however, RNase acts on the amino acid transfer RNA by modifying the specificity of its constituents, the resulting alterations of the decod-
ing system could lead to the appearance of abnormal proteins by introducing systematic errors in the ordering of the amino acids, the information carried by the deoxyribonucleic acid (DNA) remaining unaltered. It may be noted that analogous results have been obtained by the action of purine or pyrimidine analogs in other systems (Chantrenne, 1958 ; Chantrenne and Devreux, 1960; Hamers and Hamers-Casterman, 1961; Naono and Gros, 1960a,b; Bussard et al., 1960; Benzer and Champe, 1961), MATERIAL
AND METHODS
The lysogenic and sensitive strains of Bacillus megaterium used, the culture and induction techniques, and the method of phage titration have been described previously (Jeener, 1958a,b). The bovine pancreatic RNase (Sigma) is added to the cultures 20 minutes after induction, that is, at, the time when synthesis of phage protein begins. If it is added earlier, there is too great an inhibition of phage protein synthesis (Jeener, 195813)) preventing the harvesting of adequate amounts of material. The concentration of RNase used is chosen (unless otherwise specified) such
169
170
JEENER
AND VANSANTEN
that lysis is retarded by at least 2 hours and that the number of plaques is reduced 30300 times per unit weight of phage DNA. The latter is measured by analysis of the immune precipitate from a known volume of lysate. The amount of RNase required varies both with the RNase preparation and with bacterial strain; it must be determined before each series of experiments. It may be mentioned here that RNase added to an induced culture which has already lysed does not produce any of the effects to be described below. Thus RNase is totally without direct effect on complete phage particles. Ss5-labeled phage are prepared by the addition of S35-methionine (Radiochem. Centre, Amersham, England) immediately after induction of a culture, in the usual medium from which casein hydrolyzate has been omitted. The starting material for the preparation uf purified phage is a partially or totally lysed culture to which have been added lysozyme (1 mg per 100 ml) and chloramphenicol. This lysed culture is subjected to three cycles of centrifugation, alternately at 3500 g and at 65,000 g. The cfficiency of purification is controlled by electron microscopy, which shows only t’ract impurities at magnification ranging to 40,000 X. In some cases, the presence of contamination with bacterial constituents was estimated, by purification of unlabeled phage in the presence of a lysozyme lysate of sensitive S3s-labeled bacteria, to be at most 2%. Estimation of phage DNA was carried out by the method of Ceriotti (1952). To compare the ability of different phage preparations t,o adsorb onto sensitive bacteria, S3”-labeled phage was added to a growing culture in the proportion of one phage to 10 bacteria. At intervals between 0 and 10 minutes, the bacteria were centrifuged, and the serum-precipitable radioactivity of the supernatant was measured. Examination of phage with the elect,ron microscope (Siemens Elmiskop) was done after shadowing with palladium or using the phosphotungstic acid negative staining technique.
The immunological techniques used are described with the experimental results. When dispersed particles were used, the dispersion was done by treating the phage suspension in a thin-walled Lusteroid tube for 12 minutes in a sonic oscillator Raytheon, 250-watt, IO-kc, at maximum intensity. The duration of the sonic treatment was such that no longer were structures distinguishable under the electron microscope, nor did centrifugation at 65,000 g for 30 minutes produce any appreciable sedimentation of phage material. The technique chosen for the preparation of tryptic peptides is that used by Brenner et al. t.1959) when the amounts of phage proteins available are very small (denaturation at pH 2, treatment with deoxyribonuclease, digestion by trypsin in presence of 2% NH4HCOs, pH 8). The separation of peptides was carried out by chromatography and by electrophoresis on Whatman filter paper 3 MM under the conditions described by Katz et al. (1959) for the separation of tryptic and chymotryptic peptides of phage T2. RESULTS Comparison between Particles Cultivated in the Presence of RNase and Kormal Particles If RNase is added to induced cultures of B. megaterium lo-20 minutes after induction, that is, at the time when phage pyotein synthesis begins, there is a decrease both in the amount of phage produced and in the infectivity (plaques per unit weight of DNA) of the particles. Figures l-3 present the results of a particularly clearcut experiment. The number of plaques per unit weight of phage DNA decreases approximately linearly as the concentration of RNase increases. Growth of the bacteria after induction is normal until lysis, which is complete although somewhat delayed. With concentrations of RNase of 50 to 100 pg/ml, lysis becomes incomplete, the number of plaques per unit weight of phagc DNA being reduced by as much as 300 times (see below). In the above experiment the quantity of
ALTERED
PHAGE
PROTEKS
phage was measured by the amount of DNA precipitated by addition of antiserum to a known volume of lysate. In the experiments that follow, the phage particles were purified by three successive cycles of centrifugation. These preparations examined by electron microscopy contained no appreciable amounts of bacterial debris. They were immediately examined for numbers of plaques per unit weight of DNA, for the proportion of particles adsorbed onto sensitive bacteria, and for capacity to fix neutralizing antibody. The results of these tests may be summarized as follows: On the one hand, there is a reasonable parallelism between the number of plaques per unit weight of DNA and the number of particles adsorbed. For example, in three experiments in which infectivity decreased 5-, 21- and 253-fold, respectively, the proportion of adsorbed particles diminished, respectively, from 86% to
1
/
I
I
UNDER
RNASE
RIBONUCLEASE
AODED
lpglml
I
FIG. 2. Quantity of phage (dry weight of precipitate obtained with antiphage serum) and number of plaque-forming particles produced per milliliter of cultures in the presence of various concentrations of RNase (same experiment as Fig. 1). Quantity of phage and number of infectious particles are expressed as percentages of the values for a control culture without RNase.
1 ,
I .
171
ACTION
I
I
O/bg RNaso
0 10/q
”
r g ”” 030/q A20
I 0
I 10 RIBONUCLEASE
I
20 ADDED
‘jtg
I JO I ml 1
J LO
FIG. 3. Infectivity of phage produced in the presence of various concentrations of RNase (same experiment as Figs. 1 and 2). Infectiv-ity is measured by the number of plaques corresponding to the same weight of DNA precipitable by antiphage serum. b I
I
I
I
_I
FIG. 1. The optical density of lysogenic cultures induced at time zero after addition of different concentrations of RNase at the time indicated by an arrow.
20%, 90% to 5%, and 97% to nonmeasurable value. The loss of capacity to adsorb adequately explains the low infectivity of particles produced in the presence of RNase. On the other hand, there is no parallelism between the number of plaques and the capacity to fix neutralizing antibodies. As the
172
JEENER
AND
VANSANTEN
Fro. 4A FIG. 4. Preparations of phage purified by three cycles of centrifugation. cultured in the presence of RNase; C, cultured in the presence of RNase, where tails remained attached.
concentration of RNase increases, the capacity to fix neutralizing antibodies is reduced at most 3 times, whereas the number of infectious particles decreases down to l/300 the normal value. Hence, particles that are neither infectious nor able to adsorb onto sensitive bacteria are nevertheless able to fix neutralizing antibodies. Electron microscopy of particles produced in the presence of RNase usually reveals that most particles have lost the long tail that is characteristic of the phage studied (Fig. 4, A and B). We believe that the loss of the tail may result from mechanical effects during spreading and dehydration on the electron microscope grid for the following reasons. Occasionally, phage preparations whose infectivity was reduced 30 to 300 times showed a majority of particles with a tail, but the tail was pulled away from the head and attached to it by a fine filament (Fig.
A, control; 13, except)ional case
4C). In other cases, intact tails separated from the heads and clumped together were clearly recognizable; the number of tails identifiable represented 50% of the number of heads. Also, if it is assumed that the ability to fix neutralizing antibodies resides in tail constituents in the phage (as in the case for phage T2 of Escherichin coli) (Lanni and Lanni, 1953)) the purified noninfectious particles that can fix considerable amounts of neutralizing antibodies must still have a tail. Finally, the ratio of DNA to protein in purified particles was always the same in normal particles and in those cultured in the presence of RNase. Had the tails been absent, the ratio of DNA to proteins would probably have been modified significantly. Hence it may be concluded that phage cultured in the presence of RNase differ from normal phage by their inability to adsorb onto sensitive bacteria and by the lower stability of the links between head and tail.
ALTERED
PHAGE
PROTEINS
UNDER
Fm. 4B
FIG. 4C
RNASE ACTIOX
173
174
JEENER
Phage
AND VANSANTEN
proteins
added kountslmin)
FIG. 5. Antiphage serum precipitation curves for proteins of normal phage (curve 1) antI of phage grown in the presence of RNase concentrations of 40 pg/ml (curve 2) and 60 pglml (curve 3). The protein preparations had the same specific radioactivity (S”). The proteins added and precipitated were measured by their radioactivity.
Immunological Properties Compared An immunological examination of phage constituents other than those which fix neutraliaing antibody was done after sonic disruption of the particles. Phage were cultured in the presence of Ss5-methionine added at the time of induction. The phage particles were purified, sonicated, and added in increasing amounts to a constant quantity of a rabbit antiserum against purified normal phage (diluted &j-fold with 0.9% NaCl). After 2 hours at 37” and 48 hours at 4”, the precipitates were centrifuged and washed with 0.9% NaCl. The radioactivity was measured in the precipitates and in the supernatants. With disrupted control phage, maximum precipitation completely exhausted the antibody, as shown by tests on the supernatant. Hence, the sonic treatment does not destroy sponding antibodies. With proteins derived from phage cultured in the presence of increasing concentraticns of RNase, there was a progressive decrease in the maximum amount of precipitation, as shown in Fig. 5. These results indicated that the proteins of the head were quite definitely altered, like those of the tail. The progressive decrease of precipitation with increasing con-
the disappearance at unequal rates of the various reactive groups carried by the different phage proteins. As a control, absorption experiments were carried out to determine the amount of antibody fixed by equal weights of normal proteins and of proteins from phage grown in the presence of RNase. Mixtures of antiserum, appropriately diluted in 9% NaCl with increasing quantities of sonically disrupted preparations were incubated for 2 hours at 48”, then left, at 4” overnight. The precipit,ates were separated by centrifugation, and the supernatant,s were collected. Each supernatant then received a constant amount of control phage protein labeled with S:j”methionine such that 95% would be precipitated by the initial unabsorbed amount of antibody. After 2 hours at, 37” and overnight in a refrigerator, the proteins were centrifuged and washed and the radioactivity was measured in the precipitate and in the supernatant. The proportion of antigen precipitated served as a measure of the amount of the antibody that remained in the supernatant after the absorption procedure. Results are presented in Figs. 6 and 7. The antibody-fixing power of the antigens can be compared by the amounts that are needed to leave 50% of -the Ss5 unadsorbed. It is clear that the amount of phage
centrations
protein
the affinity
of their
of RNase
proteins
for the corrt-
could be explained
by
needed
to fix
a given
amount
of
ALTERED
PHAGE
PROTEINS
antibody increases with increasing concentrations of RNase present during protein synthesis. For the highest concentration of RNase that we could use, the proteins fix I1 0 78 E GE
B .s Ix .; 'd h & 21 =D' = E <.; E z %
10 9-
I
a: oyg
RNarelmt
o:6Opg
”
I
I,,,,. 0
6-
,b-
5432' %
I I I I 20 30 40 50 test
lo-20 times less antibody than equal weights of normal protein. These findings confirm that the phage head proteins have been changed considerably, since the number of antibody combining groups per unit weight of protein is greatly reduced. Test of RNase Inactivated Acid
7-
10 of
175
RNASE ACTION
c
a-
0
UNDER
phdge
protein
,
I
I
I 100
nonprecipitated
FIG. 6. Fixation of antibody by the proteins from control phage and from phage synthesized in the presence of RNase. First part : various amounts of sonic extracts from normal phage and from phage grown in the presence of RNase were added to a fixed amount of antibody. Second part: fixed amounts of S%-labeled test phage are added. The percentage of label not precipitated is estimated.
by Bromoacetic
It is important to test whether t.he RNase functions by virtue of its enzymatic activity or by some different property, for example, its basic character. Since carboxymethylation of one histidine residue causes complete inactivation of RNase without denaturing it (Barnard and Stein, 1959)) in order to study the effect of an inactive form of RNase, experiments were carried out using either normal enzyme or enzyme 90% inactivated by this method. Comparison of optical density after induction, of the amount of phage produced, of the number of plaques per unit weight of phage, and of the amount of antibody fixed per unit weight of phage protein showed that the inactivation of the RNase completely suppressed the effects of untreated RNase (see Fig. 7). There is little doubt therefore, that the effects of RNase described are due to its enzymatic activity. That RNase added to the culture medium
1 !,:
0 Org RNase/ml A 6Ojcg " 0100p "
, 1
,
(inactivate
q 1oo/Lg ‘.
; i
lo 20 30 40 % of tort phage protein
50 60 70 nonprecipitated
80
90
1010
FIG. 7. Effects of the inactivation of RNase by bromoacetic acid on the ability of phage proteins to fix antibody (see Fig. 6 legend for the technique used).
176
JEENER
AND
0
0
*
0
VANSANTEN
be profoundly altered. A study of the nature of localized changes in the proteins was undertaken to help clarify the mode of action of RNase. Fingerprints obtained by two-dimensional electrophoresis and chromatography of tryptic peptides of RNase-grown phages were different from those of control phages. Denaturation and trypsin digestion were performed under identical conditions and led to reproducible results. However, in view of the very small amount of protein available, it would seem hazardous at present to draw any conclusions as to the origin of the differences. These differences confirm, however, the abnormal character of the proteins synthesized in the presence of RNase. DISCUSSION
00 FIG. 8. Paper electrophoresis and chromatography of the peptides liberated by trypsin (continuous lines: the most clearly visible peptides obtained from normal phage protein. Dotted lines: the most clearly visible peptides from proteins of phage cultured in the presence of RNase). Electrophoresis under 2000 volts for 75 minutes at pH 3.7; chromatography in n-butanol-acetic acid-water (4: 1: 5).
can act enzymatically inside the cells of B. megaterium has been shown by Y. Robin (1962)) who observed that increasing concentrations of RNase cause a progressive decrease of the nonribosomal RNA up to 45%, ribosomal RNA and DNA remaining unaffected. No such effect was observed with enzyme inactivated by the method of Barnard and Stein. The Nature of the Modifications by the Phage Proteins
Undergone
The modified phage proteins can still aggregate and form particles. Their surface properties must therefore be fairly close to normal. Hence it seemed probable that the folding of the polypetide chains would not
It seems unlikely that RNase leads to the synthesis of abnormal proteins by modifying the information carried by the phage DNA. If that were the case, RNase would be a powerful mutagenic agent, a possibility not supported by examination of the plaques produced by the surviving phage part’icles. The fact that RNase inactivated by bromoacetic acid is altogether without effect favors the idea that it’ acts enzymatically either on the messenger RNA which directs the synthesis of phage proteins, or on the S-RNA which orders the amino acids on the messenger. The hypothesis that the RNase acts on the messenger RNA, causing formation of abnormal proteins, is doubtful; for it could only fragment the messenger, giving rise to pieces which would either be inactive or produce incomplete polypeptide chains. It is hard to imagine that such chains could fold into tertiary structures normal enough to aggregate and form particles of normal appearance. More tempting is the hypothesis that the RNase acts on the S-RNA. These nucleic acids are stable (Hoagland and Comly, 1960). During our experiments, they were constantly exposed to the attack of RNase whereas the messenger RNA, which probably has a rapid turnover (Jacob and Monod, 1961) would be less likely to be acted upon by the enzyme before it functioned as a messenger.
ALTERED
PHAGE
PROTEINS
The fact that RNase applied in viva under our conditions decreases by up to 45% the S-RNA precipitable by trichloroacetic acid (Robin, 1962) suggests that some of S-RNA molecules that are still precipitable by trichloroacetic acid are also somewhat damaged. If some of the S-RNA fragments that are preferentially produced by RNase (Rushieky et al., 1961) had new specificities, either placing their normal amino acids at abnormal points on the messenger or fixing the wrong amino acid to their normal position, it would be possible to explain the changes in immunological properties of the proteins synthesized in the presence of RNase. Then we would have to admit that the mechanism of decoding of genetic information can be modified by enzymatic activity, the character of the resulting proteins depending on the specificity of the enzyme. It would be interesting to carry similar experiments using ribonucleases of various specificities. N. DuPont-Mairesse in our laboratory carried out a series of experiments which indicated that a large part of the phage proteins synthesized in the presence of RNase are unable to assemble into phage particles (DuPont-Mairesse, 1962) but are recognizable by their capacity to combine with antiphage antibodies. In contrast, the proteins we studied could combine to form particles but had lost their reactivity with the antibodies. Thus, the action of RNase may lead to modification either of the immunologically reactive site or of the sites by which
the molecules
combine
to form
phage particles. Abnormal molecules carrying both defects, if present, would have escaped detection in our experiments. ACKNOWLEDGMENTS This work was made possible by the efficient technical assistance given by Mr. R. De Neyn. Dr. Beaufays and Dr. Vanderhaeghe gave indispensable help with the electron microscope studies. The interpretation of the results is the fruit of many discussions with Dr. De Deken-Grenson and Dr. R. Hamers. Our colleague H. Chantrenne kindly read the text and made many useful suggestions. The manuscript was translated by Miss Hell. To all of them, the authors express their warmest thanks.
UNDER
177
RNASE ACTION
The work was carried out under the association contract 016-61-10 ABIB between Euratom and the Universitd libre de Bruxelles. REFERENCES BARNARD, E. A., and STEIN, W. D. (1959). The
histidine residue in the active centre of ribonuclease. I. A specific reaction with bromoacetic acid. J. Mol. Biol. 1,339-349. BENZER,S., and CHAMPE,S. P. (1961). Ambivalent rI1 mutants of phage T4. Proc. N&t. Acad. Sci. U. S. 87,1025-1038.
BRENNER, G., STREISINGER,G., HORNE, R. W., CHAMPE, S. P., BARNETT, I,., BENZER, S., and REES, M. W. (1959). Structural components of bacteriophage. J. Mol. Biol. 1, 281-292. BUSSARD,A., NAONO,F., GROS, F., and MONOD,J. (1960). Effets d’un analogue de l’uracile sur les propriktes dune proteine enzymatique synthktisee en sa presence. Compt. rend. acad. xi. 250, 404s4051.
CERIOTTI, G. (1952). A microchemical determination of deoxyribonucleic acid. J. Biol. Chem. 198,
297303. CHANTRENNE, H.
(1958). La 8-azaguanine provoque-t-elle la formation de proteines anormales. Biochem. Pharmacol. 1,233-234. CHANTRENNE, H., and DEVREUX, S. (1960). Restauration de la synthese d’enzymes apres inhibition par I’azaguanine. &o&m. et Biophys. Acta 41,239-245. DUPONT-MAIRESSE, N. (1962). On the abnormal character of phage precursor proteins synthesized in presence of ribonuclease. Biochim. et Biophys. Acta 61, 129-134. HAMERS, R., and HAMERS-CASTERMAN, C. (1961). Synthesis by Escherichia coli of an abnormal p-
galactosidase in the presence of thiouracil.
J.
Mol. Biol. 3, 166174.
HOAGLAND,M. B., and COMLT, L. T. (1960). Interaction of soluble ribonucleic acid and microsomes. Proc. Natl. Acad. Sci. U. S. 46, 1554-1563. JACOB,F., and MONOD,J. (1961). Genetic regulatory mechanisms in the synt.hesis of proteins. 1. Mol. Biol. 3,318356. JEENER, R. (1958a). The action of ribonuclease on
sensitive
and non-induced
lysogenic
cells of
Bacillus megaterium. Biochim. et Biophys. Acta 32,99-105. JEENER, R. (1958b). The action of ribonuclease on
phage protein synthesis by an induced lysogenic Bacillus megaterium culture. Bioehim. et Biophys. Acta 32,106116. JEENER, It., DUPONT-MAIRESSE, N., and VANSANTEN, G. (1960). Proteines anormales de phages synthetisees en presence de ribonuclease. Biochim. et Biophys. Acta 45,245-255. KATZ, A. M., DREYER, W. J., and ANFINSEX, C. B.
178
JEENER
AND VANSANTEN
(1959). Peptides separation by two-dimensional chromatography and electrophoresis. J. Biol. Chem. 234,2897-2900. LANKI, F., and LANNI, Y. THBRY (1953). Antigenic structure of bacteriophage. Cold Spring Harbor Symposia Quant. Biol. 18,159-168. NAONO, S., and GROS, F. (1960a). Effets d’un analogue de base nuclkque sur la biosynthke de protknes bactbriennes. Changements de la composition globale. Compt. rend. acad. xi. 250, 35273529. NAOKO, S., and GROS, F. (1960b). Synthke par
Escherichia coli d’une phosphatase modifike en prksence d’un analogue pyrimidique. Compt. rend. acad. sci. 250,3889-3893. ROBIN, Y. (1962). Relation entre la tcneur des cellules de Bacillus megaterium cn acide ribonuckique non ribosomial et la I-itesse de synthke des protknes. Biochim. et Biopkys. Acta 55,556-557. RUSHIZKY, G. W., KNIGHT, C. A., and SOBER, H. A. (1961). Studies on the preferential specificity of pancreatic ribonuclease as deduced from partial digests. J. Biol. Chem. 236, 2732-2737.