unstable.'-3 Thus a single mrna molecule, attached to a ribosome, serves

Transcription

1 VOL. 48, 1962 BIOCHEMISTRY: TISSIl8RES AND WATSON Alexander, N. M., Anal. Chem., 30, 1292 (1958). 5 Roberts, E., and G. Rouser, Anal. Chem., 30, 1291 (1958). 6 Benesch, R., R. E. Benesch, M. Gutcho and L. Laufer, Science, 123, 981 (1956). 7Leslie, J., D. L. Williams, and G. Gorin, Anal. Biochem., 3, 257 (1962). 8 Benesch, R. and R. E. Benesch, J. Biol. Chem., 236, 405 (1961). 9 Smyth, D. G., A. Nagamatsu, and J. S. Fruton, J. Am. Chem. Soc., 82, 4600 (1960). 10 Riehm, J. P., and J. C. Speck, Am. Chem. Soc., Div. Biol. Chem., Abstracts, p. 34 C, Chicago, Sept. 3-8 (1961). Hendry, E. B., Clin. Chem., 7, 156 (1961). 12 Drabkin, D. L., Federation Proc., 16, 740 (1957). 13 Deibler, G. E., M. S. Holmes, P. L. Campbell, and J. Gans, J. Appl. Physiol., 14, 133 (1959). 14 White, A., P. Handler, E. L. Smith, and D. Stetten, Principles of Biochemistry (2d ed.; New York: McGraw-Hill Book Co. Inc., 1959), p Goldstein, J., G. Guidottig, W. Konigsberg, and R. J. Hill, J. Biol. Chem., 236, PC 77 (1961). 16 Wyman, J., Jr., J. Biol. Chem., 127, 581 (1939). BREAKDOWN OF MESSENGER RNA DURING IN VITRO AMINO ACID INCORPORATION INTO PROTEINS BY A. TISSIPRES* AND J. D. WATSON THE BIOLOGICAL LABORATORIES, HARVARD UNIVERSITY Communicated by John T. Edsall, April 3, 1962 Messenger RNA (mrna) which carries information for the synthesis of specific proteins from DNA to ribosomes, unlike ribosomal and transfer RNAs, is metabolically unstable.'-3 Thus a single mrna molecule, attached to a ribosome, serves to make possibly one, or in any case a limited number of protein molecules. It is then broken down, while new mrna molecules, made on DNA templates, function for the synthesis of new proteins. There are now indications that mrna is unstable in cell-free extracts and is broken down to acid soluble fragments.4 5 In the work presented here, the conditions for this in vitro breakdown were studied. The results show that for mrna breakdown to occur, both ribosomes and supernatant are necessary. Moreover the rate of breakdown is greatly increased by the addition of ATP and an ATP generating system. Material and Methods.-1. C14 uracil labeling of mrna: The pyrimidine requiring strain of E. coli B 148 was cultivated, and the C14 uracil pulse labeling of T2 mrna were done, essentially as previously described,5 with the exception that the cells were not starved for uracil before labeling. Growth was followed by optical density measurements, and C14 uracil was added shortly before exhaustion of the unlabeled uracil present in the medium (4,ug per ml, allowing growth to about 5 X 108 cells per ml). The cells, washed twice in M Tris-HCl ph 7.3 and M Mg++, were kept frozen. 2. Preparation of cell-free extracts, ribosomes and supernatant: E. coli B was cultivated and washed, and the crude cell-free extract was made by grinding with alumina and extracting with 3 volumes of M Tris-HCl ph 7.3 and 0.01 M Mg++, containing 5 ug deoxyribonuclease (I)Nase) per ml as described elsewhere.6 The washed cells could be kept frozen. In some experiments the extracts were fractionated into ribosomes and supernatant by 2 hr centrifugation at 100,000g. The ribosomes were washed twice in at least twice the original volume of the extract of Tris-0.01 M Mg++ mixture. The upper 2/3 of the supernatant was recentrifuged for 2 hr at

2 1062 BIOCHEMISTRY: TISSIIRES AND WATSON PROC. N. A. S. 100,000g to ensure the elimination of all ribosomes and the upper 2/3 was carefully removed to form the supernatant fraction. 3. RNA synthesis in vitro: This was carried out as described.6 The supernatant used was obtained by centrifuging an E. coli strain B extract, made without the addition of DNase, for 1.5 hr at 100,000g. The upper 2/3 was then pipetted off. It could be kept frozen. The reaction mixture contained 5 Mmoles phosphoenol pyruvate (PEP), 40,ug pyruvate kinase (PK), 0.5 ;&mole each of adenosine-5'-, cytidine-5'-, guanosine-5'-, and uridine-5'-monophosphates, M Mg++, M Mn++, M KCl, and M Tris-HCl, ph 7.4, and 0.5 ml supernatant per ml. The mixture was incubated for 9 min at 300C; 20 jug DNase per ml was added, and after one more minute at 30'C, the mixture was cooled in an ice bath. The RNA was then isolated by means of ammonium sulfate or phenol as described under Results. 4. Determination of mrna: 2.5 ml cold 5%' trichloroacetic acid (TCA) was added to the samples (usually 0.25 or 0.30 ml); the mixtures were kept in an ice bath for 20 min. They were filtered on Millipores (pore size: 0.65 M) and the precipitates were washed twice with 2.5 ml cold 5% TCA. The radioactivity on the Millipore filters was determined. 5. Amino acid incorporation: This was done as described previously.6 The reaction mixture contained 1,umole ATP, 5,mole PEP, and 40,ug PK per ml reaction mixture, with a magnesium concentration of 0.01 M. The total volume of each sample was 0.2 to 0.3 ml. With the culture medium used, consisting of mineral salts, ammonium sulfate, casamino acids, glucose, and phosphate buffer, the amino acid pool in the extract was low, and a complete mixture of all 20 current amino acids was always added in addition to C'4 alanine. The optimum concentration was found to be ,ug of each amino acid, including C14 alanine, per ml reaction mixture. These conditions were therefore used when measuring amino acid incorporation. The C'4 alanine incorporated into proteins was measured accurately after elimination of the C14 uracil label in mrna by heating in TCA. The samples were washed twice with 5% TCA, heated at 90 C for 10 min, washed again twice with 5% TCA, heated once more at 90 C for 10 min in 5% TCA, cooled, filtered on Millipores, and washed on the filters with 5% TCA. The radioactivity on the filters was determined. This procedure, more rapid than the usual procedure of Siekewitz,7 was found to give similar results in the type of experiments described here. Results.-L. Breakdown of mrna in crude extract. Breakdown of mrna in crude extract is shown in Figure 1. The extract was dialyzed for 5 hr against 0.01 M Mg++ and Tris buffer before the experiment. The reaction took place at 30 C in a total volume of 0.2 ml with 0.05 ml extract, 0.01 M Mg++ and 0.005M Tris-HCl ph 7.3. The results show that in the absence of ATP and the ATP generating system, a breakdown is observed and in 90 min 34 per cent of the counts became soluble in cold 5 per cent TCA. In the presence of ATP, PEP, and PK, the initial breakdown was more rapid. This increased breakdown slowed down with time, and after min the breakdown rate in the presence of ATP and the ATP generating system was similar to that given by the crude extract alone. 2. Ratio of mrna breakdown and amino acid incorporation in crude extracts: The incorporation- of G14 alanine into proteins was measured with the same crude extract used in the experiment reported in the preceding section. The results are given in Figure 2. The difference in the mrna breakdown, with and without ATP, PEP, and PK with time, taken from Figure 1, is also plotted and it is seen that the shape of the latter curve is similar to that of amino acid incorporation. It is clear that at any time during the reaction the ratio of mrna broken down to amino acid incorporated was roughly the same. Similar data were obtained at 24 and 370C. The crude extract used in this experiment had been dialyzed for 5 hr to decrease the size of the pool of amino acids, free nucleotides, etc. Its optical density at 260,ug was then 130, which would correspond to.5.2 mg RNA per ml, supposing

3 VOL. 48, 1962 BIOCHEMISTRY: TISSIPRES AND WATSON A FIG. 1.-Messenger-RNA breakdown in crude extract. mrna was labeled in vivo with C14 uracil, after phage T2 infection. Each sample contained 0.07 ml crude extract (CE) (about mg RNA) in a total volume of 0.2 ml, with 0.01 M Mg++ and M Tris HCl ph 7.3. ATP, phosphoenol pyruvate (PEP), and pyruvate kinase (PK) were added in amounts of 0.2 Mmole, 1 ;&mole, and 8 Mg, respectively. A mixture of 24 gg of each of the 20 amino acids was also present. Incubation was carried out at 30'C. The reaction was stopped by addition of 2.5 ml cold 5% trichloroacetic acid (TCA), and the tubes were left for about 20 min in an ice bath. The precipitates were collected on Millipore filters and washed twice with 2.5 ml cold 5% TCA. Radioactivity on the Millipore filters was measured. that all the absorbancy was due to RNA. A more correct value for total RNA would very likely be about 10 per cent lower, or 4.68 mg per ml, as some nucleotides, and also a core of DNA would probably not have dialyzed out. As 0.07 ml crude extract was present in each tube there would have been mg RNA per tube. If mrna amounts to about 2 per cent of total RNA,9 then there would be approximately 6.56,ug mrna. After 60 min incubation, 37.4 per cent of the counts of mrna became soluble in cold TCA due to the presence of ATP, PEP, and PK. This would correspond to 2.46,g mrna. For the measurement of amino acid incorporation, 16 or 24 Mug of C14 alanine and a mixture of 16 or 24 Mug of each of the other 19 nonradioactive amino acids were used per tube. In this mixture, 4.62 Mug alanine corresponded to 0.5 Mlc or 3.C6 X 105 cpm. Thus 555 cpm (Fig. 2) corresponded to 0.007,ug alanine incorporated or approximately Mug of total amino acids. The weight ratio of mrna broken down to amino acid incorporated would therefore be about 17.6/1. 3. mrna breakdown and inhibition of amino acid incorporation: Chloramphenicol (80 ug per ml) and puromycin (40 Mug per ml) had no effect on mrna breakdown (Fig. 3) although they produced 87 and 94 per cent inhibition of amino acid incorporation, respectively, with the same extract and under identical conditions. This shows that mrna breakdown is not necessarily accompanied by amino acid incorporation. The following experiment also supports this view. It is known that at low Mg++ concentration (10-4 M Mg++) amino acid incorporation amounts to only a few per cent of its value in presence of 10-2 M Mg++.8 mrna breakdown in crude extract in 10-4 M Mg++ was therefore measured. The

4 1064 BIOCHEMISTRY: TISSIJRES AND WATSON PROC. N. A. S ~~~~~~~~(A24 ip9 each amino ocid)i - _;; _ ~~~~~~~~~2000 C l U ~~~~~~~~~~~~~~~~0 C o a.a 2.Tm oreo 1 lnn icroain odtosa gvni iue1 ihete FIG. 2.-Time course of C'4 alanine incorporation. Conditions as given in Figure 1, with either 16 or 24,ug of each of the twenty amino acids, including 2 or 3 Muc C14 alanine, respectively. The samples were washed twice with 6 ml 5% TCA in the centrifuge, heated for 10 min at 900C in 3 ml 5% TCA, and centrifuged. The precipitate was washed once in 6 ml 5% TCA in the centrifuge, heated again to 90'C for 10 min in 3 ml 5% TCA, and filtered on Millipore filters, and the precipitate was again washed twice with 3 ml 5% TCA. Radioactivity on the Millipore filters was determined. The differences in the amounts of mrna broken down with and without ATP, PEP, and PK (from Fig. 1) are also plotted. 2O a _ + PEPPK+a.a.+chloramphenica1 + ATP, PEP, PK+a a.+purom FIG. 3.-Messenger-RNA breakdwn in crude extract in presence of chloramphenicol or puromycin. Conditions as in Fig. 1. Chloramphenicol and puromycin were present in amounts of 80 and 40 jmg, respectively, per ml reaction mixture.

5 VOL. 48, 1962 BIOCHEMISTRY: TISSIARES AND WATSON 1065 results obtained were similar to those shown in Figure 1: mrna breakdown took place in 10-4 M Mg++ as it did in 10-2 M Mg Breakdown of mrna in washed ribosomes: Each tube contained 0.05 ml washed ribosomes (111 Mg) in a total volume of 0.3 ml, with 0.01 M Mg++ and M Tris-HCl, ph ml cold dialyzed supernatant, prepared as described under Methods, was used per tube. ATP, PEP, and PK were added in the usual concentrations. The results (Fig. 4) show that mrna in washed ribosomes, 600E 600+ c'4 ribosomes alone] 400 _ X SU a. r + ~~~~~~~~~~ 200-; FIG. 4.-Messenger-RNA breakdown in washed ribosomes. Each sample contained 111 sg ribosomes with C14 labeled mrna (washed twice in 0.01 M Mg M Tris-HCl ph 7.3) in a total volume of 0.3 ml with 0.01 M Mg M Tris HC1 ph 7.3. Cold supernatant was added in amounts of 0.06 ml per sample, and ATP, phosphoenol pyruvate (PEP), and pyruvate kinase (PK) in amounts of 0.3 MAmole, 1.5,umole, and 12 /ug, respectively. Incubation was carried out at 30'C and the reaction was stopped by addition of 2.5 ml cold 5% TCA. The tubes were left in an ice bath for about 20 min. The precipitates were collected on Millipore filters and washed twice on the filters with 2.5 ml cold 5% TCA. Radioactivity on the Millipore filters was determined. incubated in buffer and 0.01 M Mg++, is fairly stable. The addition of supernatant alone produced about 24 per cent breakdown, while ATP and the ATP generating system by themselves produced about 50 per cent breakdown in 90 min. It is probable that the latter effect is due to the presence of enzymes from the supernatant, still bound to the washed ribosomes. It is clear that the breakdown was much greater in presence of both supernatant and ATP, PEP, and PK, particularly in the early part of the incubation. Assuming that mrna forms 2 per cent of the RNA present in the ribosomes, there was 1.4 Mg mrna per tube (111 Mxg ribosomes, and therefore 70,ug ribosomal RNA). Two washings of the ribosomes in the conditions described above had reduced the radioactivity by about 50 per cent. Thus there would be 0.7 Mug mrna per tube. Of this, about 80 per cent or 0.56 ug mrna broke down in 90 min on addition of supernatant, ATP, PEP, PK, and the amino acid mixture. Amino

6 1066 BIOCHEMISTRY: TISSIERES AND WATSON PROC. N. A. S. acid incorporation was measured in the presence of the same amino acid mixture including in addition C14 alanine, and it was found that /Ag of amino acids had been incorporated in 90 min of reaction. Thus the ratio of mrna broken down to amino acid incorporated would be about 16 to Breakdown of mrna synthesized in vitro: (a) Isolated by means of ammonium sulfate: RNA was synthesized from C14 nucleotides with 1.5 ml supernatant in a total volume of 3 ml as described under Methods. It was then cooled and fractionated in the cold by addition of neutralized saturated ammonium sulfate. The fraction precipitated between 40 and 70 per cent saturation with ammonium sulfate was collected, dialyzed for two hr against 0.01 Ml Mg++-Tris buffer and kept frozen until use. It contained over 85 per cent of the RNA synthesized in vitro, as determined by radioactivity measurements. The breakdown of RNA isolated in this way was studied at 30'C, in the presence of ATP, PEP, PK, supernatant, and ribosomes. The results are shown in Figure 5. The RNA synthesized in vitro was fairly stable even in the presence of cold ribo- +RIB C'4 RNA alone 90 - \\+ATFPPEPPK a RIB +SUP+ATPPE FIG. 5.-Breakdown of RNA synthesized in vitro, isolated by means of ammonium sulfate. The synthesis of RNA, from the four C14 nucleotide monophosphates, was carried out in a reaction mixture containing supernatant, Mn ++ and Mg++ (see text). After 9 min incubation at 30'C the reaction was stopped by the addition of 5,zg DNase per ml. It was then cooled and fractionated with ammonium sulfate. The fraction which precipitated between 40 and 70% ammonium sulfate saturation contained most of the radioactivity and was used after dialysis against 0.01 M Mg++-Tris buffer. Each sample contained 12,ug C14 RNA in a total volume of 0.3 ml, with 0.01 M Mg++ and M Tris HCl, ph j&g washed ribosomes and 0.06 ml supernatant were used per sample. ATP, PEP, and PK were added in the usual concentrations. somes or supernatant. However, on addition of ribosomes, supernatant, ATP, PEP, and PK, RNA breakdown took place. The breakdown rate was greater in the early part of the reaction. After 60 min of incubation about 50 per cent of the RNA had broken down. (b) Isolated by means of phenol: The reaction mixture, after the synthesis of RNA as above, was shaken at room temperature for 10 min with one volume of freshly redistilled phenol. The mixture was centrifuged and the water phase isolated. This treatment was repeated twice with 0.5 volume of fresh phenol. The phenol still present in the water phase was extracted with ether, and ether was removed by

7 VOL. 48, 1962 BIOCHEMISTRY: TISSIERES AND WATSON 1067 a stream of nitrogen. After two hr dialysis against 0.01 M Mg++-Tris buffer in the cold, the preparation was frozen or used immediately. The stability of the RNA prepared in this way was studied as in the preceding section, in buffer, and on addition of supernatant, ribosomes, ATP, PEP, and PK. The results of an experiment of this type, in which various amounts of ribosomes were added, were essentially similar to those of Figure 5. The breakdown with 2.25 mg of ribosomes, in a reaction mixture of 0.3 ml, was only slightly larger than with 0.90 mg of ribosomes. After 60 min of reaction, 61 per cent of the labeled RNA had become acid soluble. 6. Stability of ribosomal and transfer RNAs: In the following experiment the stability of ribosomal and transfer RNAs was studied in the presence of supernatant, ribosomes, ATP, PEP, and PK. In order to label ribosomal and transfer RNA without contamination of labeled mrna, the growing cells of the pyrimidine requiring strain were starved of uracil for 20 min; a one minute supply of C14 uracil was then added. Two min later a large excess of nonradioactive uracil was added and the cells were allowed to grow for another 30 min. The generation time in the presence of excess uracil was 40 mins. The cells were then cooled, harvested, and washed. The crude extract was prepared, the ribosomes were separated from the soluble phase by centrifugation, and ribosomal and soluble RNAs, respectively, were prepared by shaking with phenol.'0 11 The results of these experiments are shown in Figure 6. Care was taken to use amounts of ribosomal and soluble RNAs E 0 0 * z 0 a C'4Ribosomal RNA,olone 50- o C4 Ribosomol RNA +Ribosomnes 50~ o + SUP+ ATP,PEP,PK C'4 S-RNA+ Ribosomes +SUP + ATP, PEP, PK C'4 S -RNAolone I I II FIG. 6.-Stability of ribosomal and soluble RNA in presence of ribosomes, supernatant, ATP, PEP, and PK. Each sample contained C14 RNA (5.5,ug ribosomal or 5.7,ug transfer RNA) in a total volume of 0.3 ml, with 0.01 M Mg++ and M tris HC1 ph and supernatant and 700,g ribosomes were used per sample. ATP, PEP, and PK were added in the usual concentrations. comparable to those of mrna in the experiments shown in Figures 5 and 6. Consequently 5.5 and 5.7,ug of ribosomal and soluble RNAs were used in each tube, respectively. The fluctuations of the values obtained -after v ius-mes of incubation are well within the errors of the method. The stability of ribosomal and trans-

8 1068 BIOCHEMISTRY: TISSUARES AND WATSON PROC. N. A. S. fer RNAs was not appreciably affected by the addition of supernatant, ribosomes and ATP, PEP, and PK. Discussion.-When crude extracts are incubated at 30'C, mrna breaks down and the rate of this breakdown is greatly increased on addition of ATP and an ATP generating system. For this breakdown to occur both ribosomes and supernatant are necessary. It is clear that the time course of mrna breakdown corresponds rather closely to that of amino acid incorporation. The destruction of mrna may thus be the primary cause for the rapid loss of amino acid incorporation ability during incubation. After incubation has taken place and the extract has become inactive, the addition of mrna'2 or polyuridylic acidl3 results in a new burst of amino acid incorporation. Taking 2 per cent as the proportion of RNA present in the cell as mrna, it can be calculated from the data that one amino acid is incorporated into a polypeptide linkage for every four or five nucleotides released from mrna. If the amount of mrna were greater than 2 per cent, the number of nucleotides released would be greater. On the other hand, the estimation of amino acids incorporated is likely to be low, as this measurement does not take into account nonlabeled amino acids bound to transfer RNA. Note added in proof: Since recent genetic experiments suggest that the coding ratio is three to one,'4 it is likely that on the average each mrna molecule functions only once in the cell free extract. Our data do not, however, show that mrna breakdown is a necessary consequence of protein synthesis. In fact several lines of evidence suggest the contrary conclusion. First, Nirenberg and Matthaei" have reported that at least one phenylalanine residue is incorporated into peptide linkages for each uridylic acid residue in the polyuridylic acid present. This would imply that each template functions on the average three times. Second, during the synthesis of hemoglobin by the reticulocyte system, there is no indication of coupled breakdown of template RNA." Thus, it appears likely that mrna is not necessarily unstable during protein synthesis and that its short life in bacterial cells may reflect an adaptation to a need for rapidly readjusting their metabolism. It is obvious that mrna breakdown and protein synthesis can be completely dissociated. First, some breakdown occurs in the absence of an ATP generating system. Second, the increased breakdown rate, caused by the presence of ATP, PEP, and PK, is unaffected by chloramphenicol or puromycin, two powerful inhibitors of amino acid incorporation. Third, in 10-4 M Mg++, although protein synthesis is very low, mrna breakdown occurs just as well as it does in 10-2 M Mg++, the optimal concentration for amino acid incorporation. The experiments at low Mg++ concentration indicate that mrna attachntent to the ribosomes is not necessary for the breakdown to occur. The results suggest that a factor bound to ribosomes is involved. It is probable that ribonuclease plays no role, as transfer and ribosomal RNAs are not affected. We wish to thank Dr. J. W. Hopkins for his help in the preparation of RNA synthesized in vitro, Dr. P. F. Spahr for C14 nucleotides, Dr. M. Cannon for labeled ribosomal RNA, and Mrs. A. Nevins and V. Tissi~res fqr invaluable technical assistance. * Present address: Institut Pasteur, 25 rue du Dr. Roux, Paris 15e, France. 'Jacob, J., and J. Monod, J. Molec. Biol. 3, 318 (1961).

9 VOL. 48, 1962 BIOCHEMISTRY: YANKOFSKY AND SPIEGELMAN Gros, F., H. H. Hiatt, W. Gilbert, C. G. Kurland, R. W. Risebrough, and J. D. Watson, Nature, 190, 581 (1961). 3 Brenner, S., F. Jacob, and M. Meselson, Nature, 190, 576 (1961). 4 Cohen, S. S., H. D. Barner, and J. Lichtenstein, J. Biol. Chem., 236, 1448 (1961). 6 Risebrough, R. W., A Tissibres, and J. D. Watson, these PROCEEDINGS, 48, 430 (1962). 6 Tissires, A. and J. W. Hopkins, these PROCEEDINGS, 47, 2014 (1961). 7 Siekewitz, P., J. Biol. Chem., 195, 549 (1952). 8 Tissi~res, A., D. Schlessinger, and F. Gros, these PROCEEDINGS, 46, 1450 (1960). 9 Gros, F., W. Gilbert, H. H. Hiatt, G. Attardi, P. F. Spahr, and J. D. Watson, in Cellular Regulatory Mechanisms, Cold Spring Harbor Symposia on Quantitative Biology, vol. 26 (1961), p Kurland, C., J. Molec. Biol., 2, 83 (1960). 11 Tissi~res, A., J. Molec. Biol., 1, 365 (1959). 12 Gros, F., and D. Schlessinger; Tissieres, A., unpublished experiments. 13 Nirenberg, M. W., and J. H. Matthaei, these PROCEEDINGS, 41, 1588 (1961). 14 Crick, F. H. C., L. Barnett, S. Brenner, and R. J. Watts-Tobin, Nature, 192, 1227 (1961). 16 Nathans, D., G. von Ehrenstein, R. Monro, and F. Lipmann, Fed. Proc., 21, 127 (1962). THE IDENTIFICATION OF THE RIBOSOMAL RNA CISTRON BY SEQUENCE COMPLEMENTARITY, I.* SPECIFICITY OF COMPLEX FORMATION S. A. YANKOFSKYt AND S. SPIEGELMAN DEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF ILLINOIS, URBANA, ILLINOIS Communicated by H. S. Gutowsky, May 7, 1962 Despite the fact that ribosomal ribonucleic acid constitutes the bulk (85%) of cellular RNA, its mode of origin is little understood. While clearly not exhaustive, two alternatives can, at present, be entertained. One, would assume a DNAdependent reaction1 and the other would invoke a synthetic mechanism independent of DNA. The fact that the base composition of ribosomal RNA shows no tendency to correlate4' with homologous DNA is irrelevant to a choice between the two hypotheses. The presumed DNA segment involved might be so small as to constitute a statistically inadequate sample of the over-all base composition. Posing the problem in the form of these two alternatives suggests the following question, pertinent to a decision and amenable to experimental resolution: Does DNA contain a sequence complementary to homologous ribosomal RNA? An approach to questions of complementarity is in principle provided by Hall and Spiegelman's6 demonstration that specific hybrid formation can be exhibited between T2-DNA and the RNA synthesized in E. coli infected with T2. These experiments used labeled nucleic acids for ease of identification and took advantage of equilibrium centrifugation in density gradients to separate free RNA from that which hybridized to DNA. The technical difficulties inherent in using hybridization to establish the existence of complementarity between ribosomal RNA and some sequence in the DNA have already been discussed., The major complications stem from the numerology of the situation. For example, the 23S RNA component of the ribosomes is 1.1 X 106 in molecular weight, so that even if a specific complex were formed, it might