REPLICATION OF VIRAL RNA, XIV. SINGLE-STRANDED MINUS

Transcription

1 REPLICATION OF VIRAL RNA, XIV. SINGLE-STRANDED MINUS STRANDS AS TEMPLATE FOR THE SYNTHESIS OF VIRAL PLUS STRANDS IN VITRO* BY CHARLES WEISSMANN, GPNTER FEIX, HANOCH SLOR, AND ROBERT POLLET DEPARTMENT OF BIOCHEMISTRY, NEW YORK UNIVERSITY SCHOOL OF MEDICINE Communicated by Severo Ochoa, April 26, 1967 It has been reported earlier that the in vitro synthesis of infectious Qu RNA by Qua replicasel'2 is preceded and accompanied by the synthesis of single3 viral minus strands.4' This finding, as well as earlier observations with RNA synthetase, an enzyme prepared from MXIS2-infected Escherichia coli, led us to consider the possibility5' 6 that the template for the synthesis of viral plus strands might be a single minus strand, rather than some partially or entirely double-stranded structure (to be referred to simply as "double-stranded RNA" in what follows). To clarify this question it would be desirable to determine whether isolated single viral minus strands serve as template for the formation of infectious viral RNA while doublestranded RNA does not. Since appropriate preparations of minus strands were not available, the following approach was taken. It had been observed that Q, replicase, with Qq RNA (plus strands) as template, produced minus sti ands almost exclusively for a few minutes but shortly thereafter synthesized predominantly plus strands,4' 5 even when plus strands were present in excess. This finding suggested that minus strands were very efficiently utilized as a template by the replicase preparation, and that if denatured double-stranded RNA were added to the enzyme (or enzyme mixture), the minus strand rather than the plus strand might direct the formation of the bulk of the product. In this paper we show that this is indeed the case, since the product formed under the direction of denatured double-stranded RNA consists almost entirely of plus strands even in the earliest phase of the reaction, whereas the use of plus strands as template gives rise predominantly to minus strands in the same time period. Since undenatured double-stranded RNA did not stimulate RNA synthesis by Qua replicase significantly, it is likely that single minus strands play a key role in the replication of viral RNA. Preparations.-Viral RNA, labeled or unlabeled, was prepared as outlined by Feix et al.5 and pure double-stranded RANA, for use in the annlealinlg assays, by the RNase procedure of Billeter and Weissmann7 (method 2). C'4-labeled, partially double-stranded Qq RNA, for use as template, was prepared as follows. Nucleic acids were extracted from E. coli Q13 (0.75 liters of culture), infected with phage Q., and labeled with C14-uracil (0.1,ug/ml, 10 mc/mmole) from 20 to 45 minutes after infection, as previously described for "labeled double-stranded M\S2 RNA," in Billeter and Weissmann.7 Following the DNase treatment, the preparation was chromatographed on cellulose CF1 1 by the procedure of Franklin.8 The radioactive RNA eluting from the column with TSE buffer8 was lyophilized and dialyzed exhaustively against 0.5 mlim sodium ethylenediamine tetraacetic acid (EDTA), ph 7. The radioactive RNA (344 Mug, 800,000 cpm) was 78 per cent RNase-resistant; after heat-denaturation (21/2 mini at 100'C in 0.5 mm\i EDTA) this value dropped to 1 per cent. The radioactivity in minus strands, as measured by the specific dilution assay,9' 1 was 43 per cent. The labeling procedure employed in this case 1870

2 F5-Nm~e VOL. 57, 1967 BIOCHEMISTRY: WEISSMANN ET AL FIG. 1.-Sedimentation profile of C14-labeled, partially double- E stranded RNA (A) before and B. ~NA~uRED (B) after heat-denaturation. CA.C-DOUBLE-STRANDED RNA BC4-DOUBLE-STRANDED RNA, C'4-labeled RNA (3.5 ug in 10 2 {MARKER) RNA TOTAL (C"4) MU) was centrifuged through a 50 R Nose H3-MS2 (MARE RNA linear sucrose gradient as scribed in the Methods section. de- RESISAN Denatured RNA was prepared 000RNA-"')00- CSSTRND by heating a sample for 2.5 < looominus STRANDS TOTAL RNA (C) min at 1000 in 0.5 mm1 EDTA.! I/ A Aliquots al 'by\ of1' each fraction were C50 _j ; he \ 50 e A\ \ \ analyzed for acid-insoluble ra- 50iVe\ SUB < dioactivity (-0-0-) and -i, RNase- I^sSTWO>^*resistantradioactivity p7 (---) Z 0 o 0 or1minus God 1 strands (+ +) H3-abeled 1\IS2 RNA (S20, w FRACTION (number) = 278) was added to each sample as a marker prior to centrifugation (A A). did not yield uniformly labeled double-stranded RNA, so that the sedimentation profile (Fig. 1) is not necessarily representative of the total RNA distribution. Q,q replicase was purified essentially according to Pace and Spiegelman," with the slight modifications described earlier.5 An SI preparation5 with a specific activity of 6 (miumoles uridine monophosphate (UH\IP) incorporated/mg/min), devoid of nuclease activity,5 was used throughout. Other materials were from the sources indicated earlier.5' 7 12 Methods.-Incubations and isolation of RNA: All enzymatic incubations were carried out at 370, with M1gCl2, 12.8 mm; Tris-HCl buffer, ph 7.4, 84 mm\i; ATP, UTP, GTP, and CTP (one or more of which were radioactive), each 0.8 m\1; replicase SI, 80 gg/ml, and template, as specified in the figure legends. Acid-insoluble radioactivity was determined as described earlier.7 12 For further analysis of the enzymatic product, the reaction mixture ( ml) was diluted to about 1 ml with 0.15 M NaCl, 0.02 M Tris-HCl, ph 7, and sodium dodecylsulfate (to 1 %), and 50 mg of Chelex (Na+)/ml were added.5 The solution was twice extracted with one volume of phenol and then dialyzed 20 hours against 1 X SSC (three changes) and 20 hours against 0.5 mm\ EDTA (three changes). The yields of acid-insoluble product were around 75 per cent. RNase-resistant radioactive RNA was determined as described earlier.7' 12 Double isotope specific dilution assay: This assay was used to determine radioactive viral plus and minus strands.4 12 Aliquots of a mixture containing the H3- labeled sample, a small quantity of P32-labeled viral plus strands (as an internal standard), and unlabeled double-stranded Qu RNA in excess of the plus strands present in the mixture were distributed into annealing tubes. Varying amounts of unlabeled Qu RNA (from 0 to a 20-fold excess over the double-stranded RNA) were then added. The mixtures were taken to dryness, dissolved in 20,1 of 2.5 X SSC and heated three minutes at 1200 and 60 minutes at 850 in sealed tubes. The RNAase-resistant P32- and H3-radioactivities were determined and expressed as fractions of the input radioactivities (fp and fh, respectively). A plot of fh against the corresponding fp values gives a straight line, with the slope and intercept indicating the fraction of H3-radioactivity in plus strands and minus strands, respectively. A detailed description of the assay will be given elsewhere.'3 Figure

3 1872 BIOCHEMISTRY: WEISSMANN ET AL. PRoc. N. A. S. TEMPLATE: Q, RNA A TEMPLATE: DENATURED -INCUBATION: 2.5 min. DOUBLE-STR. RNA INCUBATION:25min. 0.8 i,o.4 A 0.4 _ 1 Z ~~~~~~~~~~~~~~~~~~~~~~~fh ~ ~ ~~~~~~~0602 INTERCEPT: 00 CL s~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. --\Q2 e SLOPE: MP.2 \ H3 SLPE _ d Q2 INTERCEPT: 0.74 &2- If 0L0P.6-1 s;b ca IIP>lT L ~~~~~0.2 x.h3 >_ > -7_f p32>-o C..) TEMPLATE: QA RNA TEMPLATE: DENATURED 4 INCUBATION: 40 min. DOUBLE-STR.RNA 0.8F INCUBATION:a40 min I-~~~~~~~~~ H'0.4-N 04 d le0.6 SiLOPE: SLOPE: 0.91 Eac INTERCEPT: IN ERCEPT: c0 r OA fp C Cr fp fna ~~~~~~~~~~B ~~~~~D ADDED Qn RNA(/ln g per tube) FIG. 2.-Determination of plus and minus strands in radioactive product of Q1replicase, by double isotope specific dilution assay. Samples from experiment described in Fig. 4 were extracted with phenol, dialyzed, and analyzed by double isotope specific dilution assay (see Methods). Each assay tube contained in 20 Ml of 2.5 X SSC, 1000 cpm of H-labeled product (<0.01 Mg), 2000 cpm of P32-labeled Q#s RNA (<9.1 p~g), 8 jug of 'pure double-stranded Q,6 RNA, and unlabeled Q# RNA as indicated. Mfter heating and annealing, the RNase-resistant radioactivities were determined and blank values (RNase-resistant radioactivity after heat-denaturation, 1% or less of input in all cases) were subtracted. Resulting values are expressed as the fraction of input radioactivity. Insets: the H3-values (fh) are plotted against the corresponding P32-values (fp), in order to determine the fraction of H'-radioactivity in plus and minus strands (slope and intercept, respectively, of the resulting straight line). The products analyzed were from reactions directed RNA (A and C) or denatured double-stranded RNA (B and D) after incubating for 2.5 by Q,# min (A and B) or 40 min (C and D). 2 shows the analysis of four H3-labeled enzymatic products with different ratios of plus and minus strands. Sucrose density gradient analysis was carried out by layering up to 0.15 ml of sample on a 5-ml linear 5-23 per cent sucrose density gradient containing 10 mm Tris-HCl buffer, ph 7.6, 5 mm MgCl2, 1 mm trisodium EDTA, 0.5 mm mercaptoethanol, and 100 mmi NaCi and centrifuging in the SW-65 rotor of the Spinco preparative ultracentrifuge at 0-2, for 90 minutes at 65,000 rpm. For the determination of infectious RNA14 in the enzymatic product, samples

4 VOL. 57, 1967 BIOCHEMISTRY: WEISSMANN ET AL > 4 mm. INCUBATION A 20mm. INGUBATION B FIG. 3.-Template activity of Q,, RNA (0-O) and denatured Q10 _ / 30-_double-stranded Qu RNA (0- O/ Qp RNA A+) in the Qs replicase a ^ ,, \ reaction. Incubation mixtures o DENATURED 20 DENATURED (final volume, 18 Ml) were pre- DOUBLE-STRANDED E STRANDED pared as described in the Methm E R RNA co ods section, using H3-labeled UTP (2 X 10 cpm/m~tmole), QN NT O0 Z Q R\A 101 NATIVE and the amount of template e NATiVE - DUBLE-STRANDED indicated. After incubation for _ RNA _ D'OUBLE-TRANDED * /A(A ) 4 or (B) 20 mmn at 370, I E. 0.2l l acid-insoluble radioactivity was 4 z determined. A blank (110 cpm) was ADDED RNA (,~Lg per amy) subtracted from each value. (50 pl) were diluted with 0.2 ml of 0.05 M Tris-HCl, ph 7.1 and, after addition of Chelex (Na+), extracted with phenol and ether.5 Results.-Denatured double-stranded RNA as template for Q0 replicase: The partially double-stranded Q0 RNA used in this work contains virus-specific RNA species designated as "replicative form"9' 15 and "replicative intermediate' 16 (referred to simply as double-stranded RNA in this paper). Prior to denaturation, the labeled material sedimented broadly around 14S; after heating for 2.5 minutes at 1000, in 0.5 mid EDTA, a substantial fraction of the labeled RNA and, as shown by annealing assays, about half of the labeled minus strands were found in a peak at 30S, the 820, of full-length Q# plus strands (Fig. 1). Denatured double-stranded RNA was tested for its template activity in the Q's replicase reaction. Figure 3A shows that the rate of RNA synthesis after four minutes of incubation was several times greater with denatured double-stranded RNA than with an equivalent amount of Q0 RNA as template, within a wide range of concentrations. After 20 minutes of incubation, the rate of RNA synthesis was about the same with either RNA (Fig. 3B). This is mainly due to the fact that even with saturating concentrations of Q9 RNA, the rate of RNA synthesis is relatively low at the beginning of the reaction and becomes maximal only after about 15 minutes, whereas synthesis proceeded at its highest rate at the very outset when the reaction was primed by denatured double-stranded RNA (Figs. 4 and 7). Undenatured double-stranded RNA did not stimulate RNA synthesis under the conditions of Figure 3. However, after 40 minutes incubation at 370, RNA syn- FIG. 4.-Time course of RNA synthesis with Q0 RNA or >_ denatured double-stranded Q0 RNA as template. In- 4 cubation mixtures were prepared as described in the ;04- Methods section, using H3-UTP (specific activity, 2 X 105 o O 3 Ccpm/mIAmole). The template concentration was 11 ml in all cases. The volumes of the reaction mixtures jug! were 2 and 0.7 ml when the template was Q# RNA and c 2 - DOEiNATURED denatured double-stranded RNA, respectively. Samples E2- DOUBLE- were withdrawn at the times indicated and diluted into 1 m 0,0 RNA DOUBLE- of cold 0.15 M NaCl, 0.02 M Tris-HCl buffer, ph 0U SINGLE- STRANDED 7.6. Aliquots were taken for the assay of acid-insoluble STRANDED Z - 05 RNA 0\0 RNA radioactivity; the remainder was plus and minus strands used for determination of (Fig. 5 and Table 1). The values 13 O i,, ^ -- are given as acid-insoluble radioactivity per ml of reaction mixture. Template used: none, X-X; native doublestranded Q0 RNyA, *-0; denatured double-stranded TIME OF INCUBATION (min.) Q#s RNA, *-0-; Q# RNA) 0-0.

5 1874 BIOCHEMISTRY: WEISSMANN ET AL. PROC. 1. A. S. A B - TEMPLATE: Q0 RNA (plus strands) TEMPLATE: DENATURED DOUBLE-STRANDED QR RNA o ~~~~~~~0 - u~~~~~~~~~~~~~q a. 0.U 020p a. 22j , TIME (min.) TIME (min.) FIG. 5.-Composition of the product of Q# replicase (plus and minus strands) after different times of incubation. Template: (A) Q6 RNA; (B) denatured double-stranded Q, RNA. The samples were from the experiment of Fig. 4. The analysis was carried out by the double isotope specific dilution assay (Fig. 2). Shaded areas, plus strands; crosshatched areas, minus strands. thesis set in, with apparently autocatalytic kinetics (Fig. 4). It is not clear whether the double-stranded RNA preparation was contaminated with traces of Qua RNA-- a likely possibility-or whether double-stranded RNA is in fact capable of eliciting a slow synthesis of single-stranded RNA. In either case, repeated replication of small amounts of single-stranded RNA on incubation for 40 minutes would finally result in extensive stimulation of RNA synthesis. It may be concluded that doublestranded Q# RNA in its native state has little if any template activity and that after denaturation it is more effective than Qq RNA in stimulating RNA synthesis by Q6 replicase. Nature of the product synthesized under the direction of denatured double-stranded RNA: The use of Q6 RNA as template leads to the predominant synthesis of viral minus strands during the early phase of the reaction.5 If minus strands were preferentially utilized as template in the in vitro system stimulated by denatured double-stranded RNA, then the early product should consist mainly of viral plus strands. This was found to be the case. The products synthesized by Q'6 rep- TABLE 1 SYNTHESIS OF RNA BY Q,6 REPLICASE WITH Q# RNA OR I)ENATURED DOUBLE- STRANDED RNA AS TEMPLATE ----RNA Synthesized-- - Time of Total Plus Minus RNase incubation RNA strands strands resistance Expt. no. Template (min) (Ag) (tg) (Mg) (%) 1 Qua RNA Denatured double stranded RNA The experimental conditions are described in the legend to Fig. 3. All values have been recalculated for 1 ml of reaction mixture. Template was added at 11 ;&g/ml; this was a saturating concentration for Q0 RNA but not for the sample of denatured double-stranded RNA used in this work. RNase-resistance was determined after phenol extraction.

6 VOL. 57, 1967 BIOCHEMISTRY: WEISSMANN ET AL licase, primed with either Q1B RNA or denatured double-stranded RNA, after different times of incubation, were analyzed by the double isotope specific dilution assay. Figure 2 shows the results of the analyses of the 2.5- and 40-minute products; Figure 5 summarizes the results of the entire experiment. In the case of the reaction primed with Qu RNA, the proportion of minus strands rapidly diminished from 74 per cent at 2.5 minutes to 21 per cent at 10 minutes, and 11.5 per cent at 25 minutes, while that of plus strands increased correspondingly from 10 to 72 and 88 per cent. In the reaction primed by denatured double-stranded RNA, the product initially consisted almost exclusively of plus strands; there was a small, relative increase of minus strands, from 1 to 10 per cent, as the reaction progressed. After 40 minutes of incubation, the composition of the product was the same, irrespective of the template, namely, 91 per cent plus and 9 per cent minus strands. It may be seen from Table 1 that with either template synthesis of RNA in excess of the input resulted after about 25 minutes of incubation. After four minutes of K TEMPLATE: 0$ RNA TEMPLATE: 0Q8 RNA 10 A I35 min min. 20 OTALH; 3 A INCUBATION 3 INCUBATION W 2 ~~~~40400 OAL-NI0 ~~~~~I 1P3-QoRNA IP!2Q$8RNA E A ~~ r I TEMPLATE: DENATURED DOUBLE- TEMPLATE: DENATURED DOUBLE- 20 STRANDED Q0 RNA STRANDED Q$8RNA 3.5 min. 40 min. INCUBATION 2 INCUBATION MNUS STRANDS TOTAL H3 RNA 'TOTAL H-3RNA II m and RNose ft OTLH RA0 3 RESISTANT RNA O I 0 co i Proil DePp 2 oq, RNA dobesrne 2N astmlt.teicbtoswr 5 20a D r t sp sdsrbdi h eedt 20 o i.4 FRACTION Aftero (number) -FIG. 6.-Sedimentation profile of the product of Q,0 replicase, with Q# RNA or denatured double-stranded RNA as template. The incubations were as described in the legend to Fig. 4. After 3.5 (A and C) and 40 min (B and D), respectively, samples were centrifuged through a sucrose gradient as described in the Methods section. In (A) and (B) the template was Q6 RNA, in (C) and (D), denatured double-stranded RNA. P32-labeled Q0 RNA (A-A) was added to each sample prior to centrifugation, as a marker. Q-O, Total acid-insoluble radioactivity; *-*, RNase-resistant radioactivity and minus strands.

7 1876 BIOCHEMISTRY: WEISSMANN ET AL. PROC. N. A. S. incubation the reaction stimulated by denatured double-stranded RNA resulted in the formation of about 20 times more plus strands than when Qu RNA was used as template. Sedimentation behavior of the enzymatic product: As shown earlier, the product formed during the first three to four minutes of incubation by Q6 replicase using Q9 RNA as template sediments broadly around 40S (Fig. 6A) prior to deproteinization, and consists mainly of single-stranded minus strands.5 After four to eight minutes of incubation, free plus strands, sedimenting in a narrow band at 30S, appear. In contrast to this, the product of the reaction stimulated by denatured double-stranded RNA sedimented largely at 30S after as early as 3.5 minutes of incubation, and little radioactivity was found in the 40S region (Fig. 6B). Thus, ill the reaction stimulated by denatured double-stranded RNA, the first phase of synthesis in which minus strands sedimenting at 40S are formed, is bypassed, and free, 30S plus strands are completed in this early period. After 40 minutes of incubation the product of the reaction consisted predominantly of 30S RNA, whichever the template used (Fig. 6B and D). Infectivity of the product: When Q0 replicase using Q, RNA as template synthesized RNA, an increase of infectious units could be detected only after several minutes of incubation,"7 presumably the time required to synthesize first complete minus strand templates and then full-length plus strands. If minus strands were available as template from the outset of the reaction, the latent period should be reduced to about the time necessary to complete the first plus strands. Figure 7 shows that this may indeed be the case: in the reaction primed by Qu RNA, the infectious RNA appeared after a latent period of about six minutes, whereas after priming with denatured double-stranded RNA, infectious units were synthesized after three minutes incubation (or earlier). After 30 minutes, the increase of infectivity in the Q9 RNA-primed reaction was 16-fold and that in the reaction stimulated by denatured double-stranded RNA was 32-fold. 9.0 a" A TEMPLATE: Q,0 RNA B TEMPLATE: DENATURED DOUBLE-STRANDED RNA 5 I < 0 ~~~~~~~ 4 Z0 < INFECTIOUS -3 3, CC ~~UNITS / 0 X/ ACID-INSOLUBLE X_ C- In a ACID-INSOLUBLE - 2 RADIOACTIVITY- 2 D RADIOACTIVT / 4Wi. M 0 INFECTIOUS Z X. (Al. UNITS z D < time OF INCUBATION (min.) FIG. 7.-Synthesis of infectious RNA by Q, replicase, uising Q, RNA or denatured double-stranded Q, RNA as template. The incubation mixtures (360 Al in each case) were as described in the Methods section. The labeled precursor was H3-UTP (1.2 X 10' cpm/mjimole); the template in (A) 0.06 jug Q# RNA and in (B) 1.2,ug denatured double-stranded Q, RNA. (The concentrations were chosen to give about the same number of infectious units per assay at time 0.) After incubation at 37 C for the times indicated, aliquots (5 Al) were removed for the determination of acid-insoluble radioactivity (a blank of 50 epm was subtracted from each value), and for the infectivity assay (50,ul).

8 VOL. 57, 1967 BIOCHEMISTRY: WEISSMANN ET AL Discussion.-In comparing the reactions occurring with either QO RNA or denatured double-stranded RNA as template for Q# replicase, the following main differences were noted: (a) The initial rate of RNA synthesis was greater with denatured double-stranded RNA than with Qua RNA as template. (b) The product formed during the first few minutes of the reaction consisted almost entirely of plus strands when the template was denatured double-stranded RNA, and of minus strands when it was Qq RNA. Later on, the ratio of plus to minus strand synthesis was about 10: 1 in both cases, the same value found in vivo.18 (c) As shown by sedimentation analysis, after 3.5 minutes incubation a large proportion of the product consisted of free, full-length plus strands when denatured doublestranded RNA was used as template, while in the case of Qu RNA a similar sedimentation pattern was found only several minutes later. (d) Both templates led to the formation of many times the input of infectious units, but net synthesis occurred earlier with denatured double-stranded RNA as template. Further experiments will be necessary to prove that isolated minus strands are noninfectious and can direct the synthesis of biologically active plus strands. Our findings lead us to the conclusion that when denatured double-stranded RNA is added to Qu replicase, the minus strand is preferentially utilized as template and that the product of the reaction consists mainly of full-length plus strands. Since double-stranded RNA shows little or no template activity for Qq replicase, and since single minus strands are formed in the early phase of the in vitro reaction directed by Qu RNA,4, 5 it seems likely that the template for plus strand synthesis is a single-stranded3 minus strand. We thank Mr. Morton Schneider and Mr. Winston Burrell for excellent technical assistance. We are indebted to Dr. Severo Ochoa for his constant support and valuable advice. * Aided by grants AM-01845, AM-08953, and FR from the National Institutes of Health, U.S. Public Health Service, and E. I. du Pont de Nemours and Co., Inc. 1 Haruna, I., and S. Spiegelman, these PROCEEDINGS, 54, 579 (1965). 2 Spiegelman, S., I. Haruna, I. B. Holland, G. Beaudreau, and D. R. Mills, these PROCEEDINGS, 54, 919 (1965). 3 We designate as "single" an RNA strand which is RNase-sensitive under standard assay conditions7 10 and is therefore not part of a double-helix.'0 Other definitions and abbreviations are as in previous work.5 4 Weissmann, C., and G. Feix, these PROCEEDINGS, 55, 1264 (1966). 5Feix, G., H. Slor, and C. Weissmann, these PROCEEDINGS, 57, 1401 (1967). 6 Borst, P., and C. Weissmann, these PROCEEDINGS, 54, 982 (1965). 7 Billeter, M. A., and C. Weissmann, in Procedures in Nucleic Acid Research, ed. G. L. Cantoni and D. R. Davies (New York: Harper and Row, 1966), p Franklin, R. M., these PROCEEDINGS, 55, 1504 (1966). 9 Weissmann, C., P. Borst, R. H. Burdon, M. A. Billeter, and S. Ochoa, these PROCEEDINGS, 51, 682 (1964). 10 Billeter, M. A., C. Weissmann, and R. C. Warner, J. Mol. Biol., 17, 145 (1966). 1l Pace, N. R., and S. Spiegelman, these PROCEEDINGS, 55, 1608 (1966). 12 Weissmann, C., these PROCEEDINGS, 54, 202 (1965). 13 Weissmann, C., L. Colthart, and M. Libonati, in preparation. 14 Strauss, J. H., Jr., J. Mol. Biol., 10, 422 (1964). 16 Francke, B., and P. H. Hofschneider, these PROCEEDINGS, 56, 1883 (1966). 16 Erikson, R. L., E. Erikson, and J. A. Gordon, J. Mol. Biol., 22, 257 (1966). 17 Mills, D. R., N. R. Pace, and S. Spiegelman, these PROCEEDINGS, 56, 1778 (1966). 18 Billeter, M. A., M. Libonati, E. Vifiuela, and C. Weissmann, J. Biol. Chem., 241, 4750 (1966).