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Studies on the biosynthesis of tobacco mosaic virus
J. Mol. Biol. (1974) 87, 489-503
Studies on the Biosynthesis of Tobacco Mosaic Virus VII.? Radioactivity of Plus and Minus Strands in Different Forms of Viral RNA after Labelling of Infected Tobacco Leaves M. C. KIELLAND-BRANDT Institute of Genetics, University of Copenhagen 0ster Farimugsgade 2A, DK-1353 Copenhagen K, Denmark (Received 20 December 1973, and in revised form 16 April
1974)
Tobacco leaves were labelled with tritiated uridine for 30 or 120 minutes at different times after systemio infection with tobacco mosaic virus. RNA W&B extracted and separated into three fractions: one enriched in RF (replicative form), one enriched in RI (replicative intermediate), and one containing the bulk of single-stranded RNA. Radioactivity in plus strands (viral RNA) and minus stramls (complementary RNA) W&B determined in each fraction by an isotope dilution assay. The amount of minus strands in the RF and RI fractions and the amount of plus strands in the single-stranded RNA fraction were also determined. Minus-strand synthesis was twice as high a few hours after the outbreak of visible symptoms M during the subsequent large accumulation of plus strands. At the early stage of virus production, the specific radioactivity of the minus strands was three- to fourfold that of the total RNA. Later it was about the same as that of the total RNA. As minus strands constitute a constant part of the total RNA at the later stages, this observation suggests that breakdown of minus strands is small. The specific radioactivity of minus strands was the same in corresponding RF and RI fractions. As the turn-over of minus strands appears to be small, a rapid interconversion of the two RNA types is indicated. In RF and RI the radioactivity in plus strands WM between 6 and 50 times greater than that in minus strands. The specific radioactivity of plus strands was greater in RF and RI than in the single-stranded RNA, supporting the concept that both RF and RI have a precursor role for viral RNA.
1. Introduction The replication of the RNA of RNA viruses has heen studied in most detail in the cases of the amall RNA coliphages and poliovirus. A comprehensive review has been given by Bishop & Levintow (1971). An essential part of the general scheme obtained from these studies is as follows. The viral RNA (plus strands) gives rise to the synthesis of RNA with a complementary base sequence (minus strands), which subsequently
serves as matrix for the synthesis of more viral RNA. The minus strrtnds are tied to plus strands in such a way that extraction of the RNA with phenol and detergents yields double-helical structures, the RF$ and RI. The RI is characterized by t Paper VI in this series is Nilsson-Tillgren et a2. (1974). $ Abbreviations used: RF, replicative form; RI, replicative intermediate; TMV, tobacco mosaic virus. R? 489
490
M. C. KIELLAND-BRANDT
containing base-paired as well as non-base-paired regions, whereas the RF seems to hea perfect double strand. By pulse-labelling experiments it has been shown that RI represents the structure actively engaged in replication, whereas the majority of the RF at late stages of infection is an inactive stable side-product of the replication process. Many aspects of TMV RNA replication fit into this scheme. Thus radioactivity incorporated during short periods of lahelling is found preferentially in double-
stranded RNA isolated
(Nilsson-Tillgren,
and characterized
1969), and TMV RF and TMV RI have been
(Nilsson-Tillgren,
1970; Jackson
et al., 1971).
However, work in our laboratory has also revealed differences between the replication of TMY RNA and that of poliovirus or phage RNA. Thus, except for the earliest stages of virus production, the minus strands are present as a constant fraction
of the total RNA,
(Kielland-Brandt
whereas the plus strands increase continuously
in amount
1973b). Beyond the earliest stages, the ratio of RF to RI is constant and close to one. In the RNA coliphages and poliovirus, on the other hand, the ratio of minus to plus strands remains rather constant and the ratio of RF to RI increases due to de nova synthesis of RI and turnover of RI into stable RF. For an understanding of these differences it is essential to estimate the rate of minus-strand synthesis. In the present study the radioactivities of plus and minus strands were determined in RF as well as RI after a relatively short period of labelling.
& Nilsson-Tillgren,
Furthermore,
the radioactivity
estimated, and the single-stranded activity
in non-base-paired
fraction
portions
of RI
was
was analysed for content and radio-
of plus strands.
2. Materials and Methods (a) Plant.3 and infection TMV strain tigare was used. Nkotiana tabaeum, cdtivar Samsun was grown according to Nilsson-Tillgren et al. (1969). When one of the lower leaves of this host is inoculated with TMV, a few newly expanding leaves will, 3 to 4 days later as a result of systemic infection, develop a visual symptom called vein-clearing. This symptom signifies a synchronous production of TMV in the mesophyll cells of the leaf and is distinct from the mosaic symptom appearing on leaves that expand thereafter. Vein-cleared leaves were obtained by the standardized procedure of Nilsson-Tillgren et al. (1989). All experiments were carried out on material from a single batch of plants sown and grown together, selected for similar size and appearance, and infected simultaneously. (b) Labelling Leaves were cut into strips and labelled with [5-3H]uridine Amersham) for 30 or 120 min by the method of Nilsson-Tillgren
(60 &X/ml, 5 Ci/mmol, (1969,197O).
(c) RNA eztmction RNA was extracted at 0°C by the phenol/detergent method described by KiellandBrandt & Nilsson-Tillgren (19733). Purification was as described in the same paper with the exception that DNAaae digestion was carried out for 1 h in 0.15 M-NaCl, 0.01 MM@&, 0.02 m-Tris*HCl (pH 7.2) at 37°C. (d) l%actionation of RNA The RNA was chromatographed on a cellulose column by the method of Franklin (1966) as modified by Bishop & Koch (1969). The RNA eluting in buffer containing 15% ethanol
BIOSYNTHESIS
OF TOBACCO
MOSAIC
VIRUS
491
was collected as the single-stranded RNA fraction and precipitated with 2 vol. ethanol after addition of 0.1 vol. 3 ~sodium acetate, pH 5. It was dissolved in SSC buffer (O-15 MNaCl, 0.015 ~-sodium citrate, pH 7) and istored frozen at -20°C. The RNA eluting in ethanol-free buffer was rechromatographed twice, discarding material eluting in buffer containing 16% ethanol. After addition of E.&mkhk coli RNA to 50 pg/ml, the RNA wss precipitated with ethanol as described above. After dissolution of the RNA in SSC, the RI was precipitated (together with most of the E. COGRNA) by the addition of NaCl to 1.5 M according to Bishop & Koch (1967). The RI was chromatographed once more and the RI and the RF (the supernatant in 1.5 M-NaCl) were precipitated with ethanol as described above, dissolved in SSC, and stored frozen at - 30%.
(e) Non-radioactive RP Non-radioactive RF was prepared analogously by two cycles of chromatography. Its content of minus strands was determined according to Kielland-Brandt & NilssonTillgren (1973b). (f) TMV RNA The preparation of tritiated TMV RNA hss been described previously (KiellandBrandt & Nilsson-Tillgren, 1973a). Non-radioactive TMV RNA was prepared analogously from non-labelled leaves. (g) Melting and annealing Melting (strand separation) and annealing (strand association) were performed in 4546 formamide in SSC (0.3 ml) covered with paraffi oil (Kielland-Brandt & Nilsson-Tillgren, 1973a). Melting was achieved by heating to 91°C for 3 min. Annealing wss performed at 57°C for 5, 40, or 180 min as specified. (h) Radiochemical methods Determination of the RNAase-resistant radioactivity and the total radioactivity in RNA was according to Kielland-Brandt & Nilsson-Tillgren (19730). However, the RNAase treatment was reduced to 30 min. The background (filter with no tritium) was 23 cts/min. (i) Determinatkn of RNA The concentration of the purified RNA was determined spectrophotometrically dilution in SSC taking its specific absorbancy at 260 nm ss 22.2 cm2mg-1.
after
(j ) Isotope d&h&n amay for determindion of plus strand.3 and radioactivity in plus strands in the single-stranded RNA fradona Five portions, each constituting 1140th of the radioactive sample, were melted and annealed with non-radioactive RF (0.147 fig minus strands per tube). To 4 of the tubes, different known amounts of non-radioactive TMV RNA were added prior to melting. After annealing for 180 min the RNAase-resistant radioactivity W&Bdetermined. Furthermore, two tubes were set up, each containing only one portion of the sample; one was subjected to melting and annealing whereas the RNAsse resistance of the other w&s determined without melting and without annesling. Since a large excess of plus strands was present in all tubes, the minus strands annealed completely (Kielland-Brandt t Nilsson-Tillgren, 1973a). Thus, for the 5 tubes containing non-radioactive RF the following equation can be set up: R* = p*
(B + M)
P+
A
+‘*’
where R* is RNAsse-resistant radioactivity determined after melting and annealing; P+ is the radioactivity in plus strands; B is the amount of added non-radioactive minus strands; 1cf is the amount of minus strands in the single-stranded RNA fraction (residual RF and RI);
492
31. C. KIELLAND-BRANDYP is the amount
of plus strands in the single-stranded RNA fract,ion; A is the amount of added non-radioactive plus strands ; C* is RNAaae-resistant radioactivity not due to annealed plus strands. It includes radioactivity in remaining acid-precipitable oligonucleotides and radioactivity in the minus strands of the residual amounts of RF and RI. In each series of 5 tubes B was kept constant, A was varied, and the variable Iz* w&s measured. To find P* and P it was necessary to consider C* and M. The calculation was performed as follows. (i) A preliminary estimate of P*/P was calculated from R*, B, and D* by the approximation R* = P*B/P + D*, on the tube having received non-radioactive RF but no TMV RNA. D* is the RNAase resistance of a sample determined in 0.05 x SSC; it constitutes the majority of C*. (ii) In the tube in which melting and annealing wero carried out on a sample with no additions, the RNAase-resistant radioactivity should bc: R,*
= M(l’*/P)
(iii) The RNAase-resistant radioactivity melting and annealing, can be written as Ho*
+ C*.
that
= MS0
(2)
was determined + c*,
directly,
i.o. without (3)
where S, is defined by setting MS0 equal to the RNAase-resistant radioactivity in the plus strands of the residual RF and RI present in the single-stranded fraction. AS’,,was estimated by taking a weighted mean of the corresponding value found for the RF fraction and that for the RI fraction (radioactivity in base-paired parts of plus strands per pg of minus strands, Table 5, line (D)). This is correct only if M does not include free minus strands. As found by Siegel et al. (1973) and in the present study, free minus strands are absent or low in amount. (iv) From equations (v) Equation
(2) and (3) C* and 41 were found.
(1) can also be formulated A(R*
-
C*) = P*(B
as -I- M)
-
P(R*
- C*).
Accordingly, A(R* - C*) ww;“, plotted against R * - C*. Plotting and linear regression analysis were carried out using a Hewlett-Packard Calculator 91OOA and plotter 9125B with program no. 70818. P* is found from the intercept with the ordinate by dividing by B + M. P is the slope with opposite sign. With this better estimate of P*/P, steps (iv) and (v) were repeated. This single iteration changed the results insignificantly. The or (A-l, ((R* - C*)A)-l) also offer methods for pairs of variables (A,(R* - C*)-I) finding P and P* by linear plotting. They could have been used instead.
3. Results (a) Properties
of the extracted radioactive
RNA
Tobacco leaves were systemically infected with TMIV. The procedure used leads to a fairly synchronous virus synthesis in the cells of the leaf parts showing the veinclearing symptom (Nilsson-Tillgren et aE., 1969). At three different stages of virus production these leaf parts were labelled with [3H]uridine and the RNA was extracted. At two to eight hours after vein-clearing, virus has just begun to accumulate in the cells. 26 to 32 hours after vein-clearing represents the stage where virus synthesis is at its maximum. At 74 to 80 hours the virus synthesis has again decreased (Nilsson-Tillgren et al., 1969). Some characteristics of the purified RNA are given in
BIOSYNTHESIS
OF TOBACCO TABLE
MOSAIC
493
VIRUS
1
Tritiated RNA from infected leaves Stage of virus production (time after vein-clearing) Labelling period (min)
(h)
Amount of RNA extracted (mg) Specific radioactivity (cts/min per pg) RNA&se resistance in SSC buffer (%)
2-8 30
26-32 30
7680 30
2-8 120
26-32 120
74-80 120
5.6 120 3.1
6.1 163 3.9
5.7 187 3.2
6.5 1049 2.0
7.7 994 2.1
6.4 1226 2.0
Table 1. As expected (Nilsson-Tillgren, 1969), the RNAase resistance is lower with the long than with the short labelling time. (b) Fractiondio?L of the RNA Each of the six RNA preparations presented in Table 1 was separated into a singlestranded fraction, an RF fraction, and an RI fraction as described in Materials and Methods. The recoveries of RF, RI and single-stranded RNA from the purified loaf RNA through the total fractionation are given in Table 2 ; also the RNAase resistance of the fractions. Double-stranded RNA is resistant to RNAase when tested in SSC buffer but sensitive in 0.05 x SSC. Single-stranded RNA is sensitive at both salt concentrations. It can be seen that the amount of double-stranded RNA in the singlestranded fraction was small. The recoveries given for RF and RI are based on the TABLE
2
Properties of the RNA fractions Stage of virus production (time after vein-clearing) L&belling period (min)
(h)
2-8 30
26-32 30
74-80 30
2-8 120
26-32 120
74-80 120
Single-stranded RNA fraction Recovery of RNA&se-sensitive radioactivity from the purified leaf RNA (%I RNAase resistance : in SSC (%) in 0.06 X SSC (%)
94 0.39 0.11
97 0.24 0.07
99 0.22 0.14
99 0.25 0.11
100 0.29 0.16
99 0.24 0.16
RI fraction Recovery of RI from the purified leaf RNA (bred on RNA=-resistrtnt radioactivity) (Oh) RNAase resistance : in SSC (%) in 0.05 X SSC (Oh)
64.3 54.0 0.1
49.0 44.0 0.2
37.3 27.1 0.0
45.4 29.4 0.0
60.5 29.6 0.0
46.8 23.2 0.0
RF fraction Recovery of RF from the purified lesf RNA (based on RNAase-resistant radioectivity) (Oh) RNA&we resistance : in SSC (%) in 0.06 x SSC (%)
61.6 84.7 0.0
60.3 93.6 0.3
37.3 83.0 0.1
52.1 81.4 0.1
66.7 75.7 0.1
54.1 76.7 0.1
494
M. C. KIELLAND-BRANDY?
assumption that they have the same recovery when they are chromatographed together. As will be seen below (Table 5, line (G)) the RF to RI ratios calculated from these recoveries (and the minus-strand content of the fractions) are similar to those found earlier (Kielland-Brandt & Nilsson-Tillgren, 19733). (c) Virus-specific RNA ila the RF and RI )wGo~zs The RNAase resistances given in Table 2 indicate that the RF fractions are not radiochemically pure, since TMV RF has been prepared earlier that was more resistant to RNAase (Nilsson-Tillgren, 1970; Kielland-Brandt $ Nilsson-Tillgren, 1973a). In order to find out how much of the RNAase-sensitive radioactivity in the RF and RI fractions (Table 2) resided in virus-specific RNA, portions of the fractions were annealed together with melted non-radioactive RF (Table 3). Since the difference between the two columns is insignificant, it appears that the RF and RI fract,ions did not contain detectable amounts of labelled virus-specific single-stranded RNA. It was ascertained that radioactivity in single-stranded RNA would anneal in this particular experiment by annealing, in parallel, tritiated TMV RNA with the melted non-radioactive RF. Thus little-probably less than 20a/o-of the radioactivity in the RI molecules seems to be present in the single-stranded regions. The RNAase-sensitive material in the RF and RI fractions must be due to host RNA not removed by the fractionation. TABLE
3
Virus-specijc RNA in the RF and RI fractions Stage of virus production (time after vein-clearing) (h) 2-8 2-8 26-32 26-32 74430 74-80 2-8 2-8 2632 26-32 74-80 74-80
Labelling period WN 30 30 30 30 30 30 120 120 120 120 120 120
RNAase-resistent Fraction
RI RF RI RF RI RF RI RF RI RF RI RF
In sample
287 509 504 880 232 686 1785 2478 2691 3977 1913 3319
radioactivity (cts/min) After annealing with melted, non-radioactive RF? 285 621 611 847 242 662 --t 2497 2811 4023 1886 3242
t Non-radioactive RF (1.28 pg minus strands per ml) was melted and quickly oooled in a - 16’C bath. 100 4 of this melted RF was immediately added to a portion of each fraotion, and the mixtures were subjected to annealing conditions for 40 min before determination of RNAaseresistant radioactivity. $ Not determined.
(d) Radioactivity in plus and minus strands of RF and RI Samples of the six RF fractions and the six RI fractions were melted and annealed with (i) different amounts of non-radioactive TMV RNA, and (ii) different amounts of tritiated TMV RNA. The first procedure follows the specific isotope dilution assay
radioactivkyt
(time
neither
(iii)
melting
127.7
13.3
141.0
“77.0
14.5 124 14.4 12.6 24.1
2-R 30 RI
“40.5
13.5 244.3
12.2
368.3 ‘86,5
250.3
13.5 46.5
16-4
11.7 9.2
2632 30 RI
“54.0
14.8 13.6 13.3 13.7 33.8
“- 8 30 RF
423.3
13.3
436.6
403.2
11.7 10.2 14.0 21.6 70.6
26-32 30 RF
and annealing
163.5
115.3
6.3
121.6
277.7
5.8
283.5
294.6
6.7 7.9 35.7
4.9 5.0
RF
RI
7.8 6.4 3.9 7.7 12.2
7680 30
7P80 30
776.0
126.4
902.4
387.6
‘00.5
119.9
134.7 127.5 128.2
RI
2-8 120 RI
RF
396.9
1051.7
129.6
1181.3
1258.2
75.8
1334.0
649.6
74.4 73.0 83.4 87.4 288.7
26-32 120
2-8 120
127.7 131.3 133.7 131.6 220.5
of the RF and RI fractions
4
1864.0
72.0
1936.0
688.2
88.5 76.3 408.0
69.1
76.2
RF
26-32 1’0
874.0
51.5
9PB.Q
421.0
1511.5
51.4
1562~9
510.7
51.9 48.1 58.1 60.8 279.4
RF
RI
48.6 62.1 53.9 55.3 146.2
74-W 120 74-80 120
t Cts/min per sample. Each sample was 1/30th of the fraction. The values have been correct,ed by subtracting the RNAase-resistant radioactivity of an equal sample determined in 0.05 x SSC. It was between 0 and 2 cts/min. $ Bnnealing for 5 min. The rest of the Table contains values determined after 180 min annealing. $ Four different amounts (50, 10.3, 3.5 and 1.2 pg) of tritiated TMV RNA of a specific radioaotivity of 7613 cts/min per pg. The RNAase-resistant radioactivity was corrected by subtracting O.OZ”h of the radioactivity in the tritiated TMV RNA, since that was the RNAese resistance of this RSA. Even t,he lowest amount of added tritiated TMV RNA represents a large excess of plus strands and therefore the four values should be equal. The average in given.
of plus
nor annealing
melting and annealing TMV [3H]RNA $
Estimated radioactivityt in minus strands in base-paired parts strands
after with
(ii)
11.1 /%t 0.37 fig
370 /& 11.1 N
(i) after melting and annealing with non-radioactive TMV RNS, 370 Pg
RNAase-resistant
Stage of virus production after vein-clearing) (h) Labelling period (min) Fra&ion
Jleltiqy
TABLE
496
hf. C. RIELLAND-RRANDT
of Weissmann et al. (1964) and the second the isotope dilution assay of KiellandBrandt t Nilsson-Tillgren (1973b). The RNAase-resistant radioactivity determined after melting and annealing is given in Table 4. (i) Melting and annealing with non-radioactive tobacco mosaic viru.s RNA At the two highest concentrations of plus strands, 370 pg and 11.1 pg per tube of 0.3 ml (Table 4), annealing should be practically complete after the first five minutes, as the rate constant is about 25 mg-lmin-lml (Kielland-Brandt t Nilsson-Tillgren, 1973a). 180 minutes of annealing give similar or slightly higher values, and not significantly lower values than an annealing period of five minutes. Therefore the decrease in radioactivity conferred by the addition of TMY RNA cannot be due to nuclease contamination but is a real competition phenomenon. At both concentrations practically the same RNAase-resistant radioactivity was obtained, showing that the non-radioactive plus strands competed all radioactive plus strands out. When 0.37 rg non-radioactive plus strands is added, the annealing should also be practically complete after the 180 minutes. This amount appeared to be insufficient to compete all radioactive plus strands out. To get the best estimate of the amount of radioactivity in minus strands, the RNAase-resistant radioactivity was plotted against the reciprocal of the amount of added non-radioactive plus strands. By extrapolation to the ordinate we obtained the radioactivity in minus strands given in Table 4. Samples of the fractions were also melted and annealed together with 0.037 tug non-radioactive TMV RNA and with no TMV RNA. Still more RNAaseresistant radioactivity than with the addition of 0.37 pg TMV RNA was the result (data not shown). The radioactivity in base-paired parts of plus strands (Table 4) is found by subtracting the radioactivity in minus strands from the RNAase-resistant radioactivity of an untreated portion of the fraction. This gives an error for the RI fraction if parts of the minus strands are non-base-paired. However, the error will be small, since so little of the radioactivity is in minus strands, From the data in Table 4 it was possible to estimate the total radioactivity in plus strands per sample (cf. formula (6) of Weissmann et al., 1964). It was found that the total radioactivity in plus strands did not differ significantly from the radioactivity in base-paired regions of plus strands. This result is in accordance with the data in Table 3. (ii) Melting and annealing with trztiated tobacco mosaic virus RNA The RNAase-resistant radioactivity after melting and annealing with tritiated TMV RNA is given in Table 4. After subtraction of the radioactivity in minus strands the value was divided by the specific radioactivity of the added tritiated TMV RNA, whereby the amount of minus strands per sample was obtained (Table 5, line (A)). From these values and additional data of Table 4, the specific radioactivity of minus strands, as well as the radioactivity in plus strands per /*g of minus strands, was calculated. The results are given in Table 5. It can be seen that the specific radioactivity of minus strands in each RI fraction is almost the same as in the corresponding RF fraction (line (B)). The specific radioactivity of minus strands is threeto fourfold that of the total leaf RNA at the earliest stage of virus production, but later it is about the same as that of the total leaf RNA (line (C-)). The radioactivity in plus strands relative to the amount of minus strands is somewhat, larger in RF
9.6
(E) Radioactivity in base-paired parts of plus strands relative to that in minus strands 0.60 0.79 250
(G) Amount of minus st~randa in RF divided by that in RI
(H) Cts/min in minus strand3 per mg of total RNA
pg of minus strands per mg of total RSAk
3778
(D) Cts/min in base-paired parts of plus strands per rg of minus strands:
(F)
3.3
(C) Specific radioactivity of minus st,rands relative to that of the tot,el RNA
30.2
33*8
17.8
7964
3.7
447
“-8 30 RF
Z-8 30 RI
394
of minus strands
of minus st.rands per sample
(time after
(B) Specific radioactivity (cts/min per pg)
(A) Nanograms
Stage of virus production vein-clearing) (h) L&belling period (mm) Fraction
20.0
5154
1.6
257
47.4
26-32 30 RI
230
0.89
0.90
31.8
8156
1.6
256
51.9
26-32 30 RF
.J nm/ T1.t.u of nai~,w .strand.s and radioactivity
(Oaq
170
47.9
7213
0.8
151
38.5
7P80 30 RF
(1.97)
18.3
(5883)
(1.7)
(321)
(19*6)
74s”so 30 R.1
6.X
0.89
0.67
8.1
29,540
3.5
3638
35.6
2-8 120 RF
in RF ad
2439
22,360
3.5
3643
34*7
‘-8 120 RI
in plus and rninu.s strands
TABLE 6
l&6
0.9
878
82,O
26-32 120 RF
91G
O.Y9
5.9
72.i30
0.9s
16,470
1.0
992
76.4
26-32 120 RI
RI
17,o
17,770
0.9
1047
49.2
74-80 120 RI
960
1.07
1.02
29.4
24,740
0.7
841
61.1
7680 120 RF
498
M. C. KIELLAND-BRANDT
than in RI; no change with stage of virus production is evident (line (D)). As a consequence, the ratio of label in plus strands to label in minus strands increases with time after infection and is greater in RF than in the corresponding RI (line (E)). From the recoveries during fractionation (Table 2) and the data of line (A) (Table 5), the amount of minus strands relative to the total amount of leaf RNA has been calculated (line (F)). Also the ratio of RF to RI is given (line (G)) as well as the radioactivity incorporated into minus strands divided by the amount of leaf RNA (line (H)). The RI values for the 30-minutes labelling at 74 to 80 hours after vein-clearing fall clearly outside the rest of the data: (i) in contrast to the other five labellings, the specific radioactivity of minus strands in RI is twice that in RF, though this is possibly not significant because of the low number of counts. (ii) The amount of minus strands is somewhat lower than in the corresponding 120-minutes labelling and lower than found by Kielland-Brandt t Nilsson-Tillgren (19733). (iii) The ratio of RF to RI is twice that found for the other labellings and that found by KiellandBrandt & Nilsson-Tillgren (19733). All three discrepancies are eliminated by assuming that the amount of minus strands in the RI fraction has been underestimated by a factor of two, because of some error made in the experiment. This possibility is indicated by the brackets in the Table. Accordingly, disregarding these values, the amount of minus strands was O*1o/oof the total RNA at the late stages and a little lower at two to eight hours after vein-clearing (line (F)). The ratio of RF to RI was around one at all three stages (line (G)). Line (H) indicates a preferential Rynthesis of minus strands at two to eight hours after vein-clearing. (e) Analysis of the single-stranded RNA fractions The single-stranded fractions were analysed for their content of plus strands and the radioactivity in the plus strands by the isotope dilution assay described in
50 R*-C*(cls/min)
FIG. 1. Determination of plus strands and radioactivity in plus strands in the single-stranded fraotions after 120 minutes of labelling. The isotope dilution assay used is desoribed in Materials and Methods. Abscissa: annealed radioaotivity in plus strands (R* - C*). Ordinate: amount of added non-rsdioactive TMV RNA times annealed radioaotivity in plus strands (A(R* - C*)). The amount of plus strands per sample is found 88 the slope with opposite sign. The radioactivity in plus strands per sample is found by dividing the intercept with the ordinate by the amount of minus strands per tube. --O-O--, --e-m-, 26 to 32 h after vein-clearing; -A-A-, 74 to 80 h 2 to 8 h after vein-clearing; after vein-clearing (the skew point is not included in the linear regression). The baokground, C*, was 246, 261, and 273 ots/min, respeotively. The correlation coefficient was -0.991, -O*QSS and -0.999, respectively.
BIOSYNTHESIS
OF
TOBACCO TABLE
MOSAIC
499
VIRUS
6
Xingle-stranded RNA fra&imLs, I20 minutes lube&g Stage of virus production
(time after vein-clearing)
(h)
Retio of plus strands to total RNA Retio of radioclctivity in plus strands to thst in the totd RNA Specific radioactivity of plus strands (cts/min per pg)
2-a
26-32
74-80
0.023
0.098
0.213
0.23 10,200
0.39 3960
0.27 1640
Materials and Methods. The data are given in Figure 1 and the results in Table 6. For the 30-minutes labelling the background of radioactivity in oligonucleotides p*, see Materials and Methods) was too large to allow any reasonable calculations, so only data after 120 minutes of labelling have been used. Table 6 shows that a massive plus strand synthesis was taking place during the three-day period investigated. From the data of Tables 5 and 6 were calculated the radioactivity in plus strands per pg of minus strands, the radioactivity in minus strands per pg of plus strands, and the ratio of plus to minus strands (Table 7). The radioactivity incorporated into plus strands per pg of minus strands was about the same at the three stages of virus production, whereas that incorporated into minus strands per pg of plus strands decreased more than 20 times. The ratio of plus to minus strands was increasing with time after infection. TABLE
7
Radioactivity in plus and minus strands after 120 minutes labelding Stage of virus production
(time after vein-oloaring)
(h)
Cts/min incorpormed into plus strands per pg of minus strands Cts/min incorporated into minus &rends per pg of plus strands Ratio of plus to minus &ends
2-8
26-32
74-80
348,000
394,000
321,000
107 34
9.4 100
4.6 208
By subjecting samples of the single-stranded fractions to annealing conditions together with tritiated TMY RNA without prior melting, no free (single-stranded) minus strands could be detected (data not shown). However, apart from the samples from two to eight hours after vein-clearing, this experiment was not very sensitive because of the presence of a large amount of plus strands with a low specific radioactivity.
4. Discussion (a) The jractionation Tables 2 and 3 show that the RI fractions contain a great deal of-and the RF fractions some-radioactive host RNA. The fourth chromatographic run of RI only changed the RNAase-resistant radioactivity a little (data not shown). In
500
M. C. KIELLAND-BRANDY-
separating RNA extracted from Sendai virus-infected cells, Portner & Kingsbury (1972) experienced similar difficulties with cellulose columns. The level of TMV sequences and its complementary sequences in RNA from uninfected plants is so low (Kielland-Brandt & Nilsson-Tillgren, 19733) that the incompleteness of the fractionation is negligible in the present context. The single-stranded fractions contained small amounts of RF and RI, which could probably have been removed by repeated chromatography. It was preferred to determine their amount and correct for them. (b) Amounts of plus arm! minus strands The data on amounts of minus strands and their distribution between RF and RI (Table 5) are in accordance with earlier determinations (Kielland-Brandt & NilssonTillgren, 19733). Determination of plus strands (Table 6) gave two to three times higher values than were obtained earlier, but the kinetics of their appearance confirm our observation that the ratio of plus strands to total RNA is steadily increasing whereas the ratio of minus strands to total RNTA soon reaches a constant (and low) level. The ratio of plus to minus strands (Table 7) corresponds well to that found in leaf homogenates (Nilsson-Tillgren et al., 1974) from plants grown during the same months. The differences in plus-strand content may therefore be due to small variations in the growth conditions. Kielland-Brandt & Nilsson-Tillgren (19736) found relatively large differences in plus-strand content when different batches of plants were analysed. The present experiments do not distinguish molecules of different size. Thus the TMV-induced low molecular weight RNA (Jackson et al., 1972) will simply be measured as plus-strand material (Siegel et al., 1973). However, the low molecular weight RNA represents only a minor part of the plus-strand material (Jackson et al., 1972). Most, if not all, minus strands were present in the double-stranded forms. This is in accordance with the results of Siegel et al. (1973) who did not find any free minus strands in an electrophoretic analysis. The majority of plus strands was in the single-stranded fraction. It was not possible to measure the amount of plus strands in the RI and RF fractions directly but the data on non-base-paired radioactivity in Table 3 indicate that it amounts to no more than a few times the amount of minus strands. (c) Asymmetry of lubdling of the double-stranded fornzs On the basis of annealing experiments Siegel et al. (1973) concluded that after two hours labelling the RF contains about equal radioactivity in plus and minus strands, As they point out, this is an unexpected finding. However, it was not ascertained that in the annealing with non-radioactive TMV RNA all radioactive plus strands were competed out; neither was it shown that the annealing with non-radioactive double-stranded RNA was complete. Earlier analyses of Nilsson-Tillgren revealed a pronounced asymmetry in the labelling of the double-stranded forms (cf. Nilsson-Tillgren, 1970). In the present study this asymmetry of labelling has been confirmed. Even after two hours of labelling the ratio of radioactivity in the plus strands to that in the’minus strands was between 6 and 29 depending on the stage of infection.
BIOSYNTHESIS
OF TOBACCO
MOSAIC
VIRUS
601
(d) Role of the double-stranded form It is well established that RF and RI have a precursor role for TMV RNA (NilssonTillgren, 1969,197O; Jackson et al., 1971,1972). The fact that the plus-strand material in RF and RI has a higher specific radioactivity than has the single-stranded TMV RNA (Tables 5 and 6) gives strong support to this notion. It is not known whether TMV RF and TMV RI are double-helical in viva. (e) Eficiency
of labelli’ng
The capacity of the cells for uridine uptake and the sizes of their nucleic acid precursor pools need not be the same at different stages after infection. Therefore, deductions from radioactivity to rate of synthesis in the comparison of different stages require a measure for the efficiency of labelling. The best measure in the present experiments is the specific radioactivity of the plus strands in RF. It should be equal to the specific radioactivity of the nucleic acid precursor pool shortly before termination of the labelling period. The specific radioactivity of plus strands in RF did not vary much with the stage of virus product’ion (Table 5, line (D)). (f) Rate of synthesis of plus strands In Table 7 it is seen that the radioactivity incorporated into plus strands per pg of minus strands was about the same at the three stages investigated. Since the efficiency of labelling was also about equal, the number of plus strands synthesized per minus strand per unit of time should be about the same. By dividing the radioactivity in plus strands per pg of minus strands by the specific radioactivity of plus strands in RF one obtains a minimum estimate of plus-strand initiations at a given minus strand during the two hours. It is found t,hat the average time between successive initiations at a given minus strand was maximally, ten minutes at 2 to 8 hours, seven minutes at 26 to 32 hours, and nine minutes at 74 to 80 hours after veinclearing. For other RNA viruses higher, as well as a lower, frequency of initiation has been indicated. Thus, since a poliovirus plus strand is completed in about one minute (Girard et al., 1965) and poliovirus RI contains several nascent plus strands (Baltimore, 1968), the average time interval between successive plus-strand initiations at a given minus strand should be less than one minute. Turnip yellows mosaic virus plus strands, on the other hand, have been proposed to be synthesized with the rate of one strand per hour at each site of synthesis in the cell (Ushiyama & Matthews, 1970). (g) Rake of synthesis of minus strands
When minus-strand synthesis is measured relative to the total amount of RNA or to total RNA synthesis, for the 120-minutes labelling it is about twice as high shortly after vein-clearing than at the two later stages investigated (Table 5, line (H)). For the 30-minutes labelling it is a little different, but with the low number of counts t,his is probably not significant. The specific radioactivity of the minus strands was, at the early stage, three- to fourfold that of the total RNA but at the later stages about the same as that of the total RNA. From these observations two conclusions can be drawn. First, there is a preferential synthesis of minus strands at or shortly after vein-clearing. Even on a per leaf basis the synthesis of minus strands must be higher at t’wo to eight hours after vein-clearing
602
M. C. KIELLAND-BHANDT
than at the two later stages. Second, the minus strands must be rather stable, at least at the later stages of virus production. This is deduced from the fact that at these stages the minus strands constitute a constant part of the total RNA and have about the same specific radioactivity as the total RNA. Therefore the relative rate of breakdown of minus strands can not be greater than the relative rate of breakdown of the total RNA. When minus-strand synthesis is measured relative to the amount of its template, plus strand (Table 7), a pronounced decrease with time is observed. The removal of plus strands by incorporation into virions is probably a major reason for this, but other mechanisms may aot as well. (h) Functions
of RF and RI
For the distinction between RF and RI their different solubility in 1.6 M-NaCI has been used in the present study. Nilsson-Tillgren (1970) has previously characterized in this laboratory TMV RF and RI prepared with the methods used here by sedimentation analysis in sucrose gradients, and demonstrated that precipitation with NaCl separates the double-helical RNA molecules with complete base-pairing from those containing single-stranded regions. As the specific radioactivity of minus strands is the same in RF and RI (Table 5, line (B)) and minus strands are not extensively broken down, a rapid interconversion of the two double-helical RNA forms during replication is indicated, in contrast to the situation in phage MS2 or poliovirus. Radioactivity in base-paired parts of plus strands per pg of minus strands was consistently higher in RF than in RI (Table 5, line (D)). Furthermore, no significant radioactivity was found in single-stranded viral sequences in the RI fractions (Table 3). These two results point to the possibility that the single-stranded regions of RI are minus-strand regions rather than plus-strand regions. This possible difference between the structure of TMV RI and phage MS2 RI is under further investigation.
5. Conchdons Minus strands are synthesized at the highest rate at vein-clearing or shortly after. Thereafter, during the main accumulation of plus strands, the minus strands arc synthesized at a twofold lower rate. As shown earlier, the amount of minus strands remains at O*1o/oof the total RNA during this period of time. Breakdown of minus strands is small. Most if not all minus strands are present in completely (RF) or partially (RI) double-stranded structures after the extraction. RF and RI are probably interconverted in the process of plus-strand synthesis. The precursor role of RF and RI for TMV RNA has been confirmed.
I am indebted to Dr T. Nilsson-Tillgren for introducing me to the study of TMV BNA replication and for his steady help and advice. Further, I wish to thank Professor D. von Wettstein for support and encouragement and Miss J. Verhein Hansen for skilful technical assistance. Financial support was provided by grant no. GM10819 from the U.S. Public Health Service, National Institutes of Health and the Carlsberg Foundation to Professor D. von Wettstein.
BIOSYNTHESIS
OF TOBACCO
MOSAIC
VIRUS
503
REFERENCES Baltimore, D. (1968). J. Mol. Biol. 32, 359-368. Bishop, J. M. & Koch, G. (1967). J. Biol. Chem. 242, 17361743. Bishop, J. M. & Koch, G. (1969). Virology, 37, 521-534. Bishop, J. M. & Levintow, L. (1971). Progr. Med. Viral. 13, 1-82. Franklin, R. M. (1966). Proc. Nat. Acad. Sci., U.S.A. 55, 1504-1511. Girard, M. P., Baltimore, D. & Darnell, J. E. (1965). Fed. hoc. Fed. Amer. Sot. Erp.
Biol.
24, 379.
Jackson, A. O., Mitchell, D. M. & Siegel, A. (1971). Virology, 45, 182-191. Jackson, A. O., Zaitlin, M., Siegel, A. t Francki, R. I. B. (1972). V+roZogy, 48, 655-665. Kielland-Brandt, M. C. & Nilsson-Tillgren, T. (1973a). Mol. Gen. Genet. 121, 219-228. Kielland-Brandt, M. C. & Nilsson-Tillgren, T. (19735). Mol. Cen. Genet. 121, 229-238. Nilsson-Tillgren, T. (1969). Mol. Gen. Genet. 105, 191-202. Nilsson-Tillgren, T. (1970). Mol. Gen. Genet. 109, 246-256. Nilsson-Tillgren, T., Kolehmainen-SevBus, L. & von Wettstein, D. (1969). Mol. Gen. Genet. 104, 124141. Nilsson-Tillgren, T., Kielland-Brandt, M. C. & Bekke, B. (1974). 1cfoZ.Gen. Genet. 128, 157169. Portner, A. & Kingsbury, D. W. (1972). Virology, 47, 711-725. Siegel, A., Zaitlin, M. & Duda, C. T. (1973). ViroZogy, 53, 75-83. Ushiyama, R. & Matthews, R. E. F. (1970). V’iroZogy, 42, 293-303. Weissmann, C., Borst, P., Burdon, R. H., Billeter, M. A. & Ochoa, 8. (1964). Proc. Nat. Acad. Sci.,
1T.S.A. 51, 890-897.
Studies on the Biosynthesis of Tobacco Mosaic Virus VII.? Radioactivity of Plus and Minus Strands in Different Forms of Viral RNA after Labelling of Infected Tobacco Leaves M. C. KIELLAND-BRANDT Institute of Genetics, University of Copenhagen 0ster Farimugsgade 2A, DK-1353 Copenhagen K, Denmark (Received 20 December 1973, and in revised form 16 April
1974)
Tobacco leaves were labelled with tritiated uridine for 30 or 120 minutes at different times after systemio infection with tobacco mosaic virus. RNA W&B extracted and separated into three fractions: one enriched in RF (replicative form), one enriched in RI (replicative intermediate), and one containing the bulk of single-stranded RNA. Radioactivity in plus strands (viral RNA) and minus stramls (complementary RNA) W&B determined in each fraction by an isotope dilution assay. The amount of minus strands in the RF and RI fractions and the amount of plus strands in the single-stranded RNA fraction were also determined. Minus-strand synthesis was twice as high a few hours after the outbreak of visible symptoms M during the subsequent large accumulation of plus strands. At the early stage of virus production, the specific radioactivity of the minus strands was three- to fourfold that of the total RNA. Later it was about the same as that of the total RNA. As minus strands constitute a constant part of the total RNA at the later stages, this observation suggests that breakdown of minus strands is small. The specific radioactivity of minus strands was the same in corresponding RF and RI fractions. As the turn-over of minus strands appears to be small, a rapid interconversion of the two RNA types is indicated. In RF and RI the radioactivity in plus strands WM between 6 and 50 times greater than that in minus strands. The specific radioactivity of plus strands was greater in RF and RI than in the single-stranded RNA, supporting the concept that both RF and RI have a precursor role for viral RNA.
1. Introduction The replication of the RNA of RNA viruses has heen studied in most detail in the cases of the amall RNA coliphages and poliovirus. A comprehensive review has been given by Bishop & Levintow (1971). An essential part of the general scheme obtained from these studies is as follows. The viral RNA (plus strands) gives rise to the synthesis of RNA with a complementary base sequence (minus strands), which subsequently
serves as matrix for the synthesis of more viral RNA. The minus strrtnds are tied to plus strands in such a way that extraction of the RNA with phenol and detergents yields double-helical structures, the RF$ and RI. The RI is characterized by t Paper VI in this series is Nilsson-Tillgren et a2. (1974). $ Abbreviations used: RF, replicative form; RI, replicative intermediate; TMV, tobacco mosaic virus. R? 489
490
M. C. KIELLAND-BRANDT
containing base-paired as well as non-base-paired regions, whereas the RF seems to hea perfect double strand. By pulse-labelling experiments it has been shown that RI represents the structure actively engaged in replication, whereas the majority of the RF at late stages of infection is an inactive stable side-product of the replication process. Many aspects of TMV RNA replication fit into this scheme. Thus radioactivity incorporated during short periods of lahelling is found preferentially in double-
stranded RNA isolated
(Nilsson-Tillgren,
and characterized
1969), and TMV RF and TMV RI have been
(Nilsson-Tillgren,
1970; Jackson
et al., 1971).
However, work in our laboratory has also revealed differences between the replication of TMY RNA and that of poliovirus or phage RNA. Thus, except for the earliest stages of virus production, the minus strands are present as a constant fraction
of the total RNA,
(Kielland-Brandt
whereas the plus strands increase continuously
in amount
1973b). Beyond the earliest stages, the ratio of RF to RI is constant and close to one. In the RNA coliphages and poliovirus, on the other hand, the ratio of minus to plus strands remains rather constant and the ratio of RF to RI increases due to de nova synthesis of RI and turnover of RI into stable RF. For an understanding of these differences it is essential to estimate the rate of minus-strand synthesis. In the present study the radioactivities of plus and minus strands were determined in RF as well as RI after a relatively short period of labelling.
& Nilsson-Tillgren,
Furthermore,
the radioactivity
estimated, and the single-stranded activity
in non-base-paired
fraction
portions
of RI
was
was analysed for content and radio-
of plus strands.
2. Materials and Methods (a) Plant.3 and infection TMV strain tigare was used. Nkotiana tabaeum, cdtivar Samsun was grown according to Nilsson-Tillgren et al. (1969). When one of the lower leaves of this host is inoculated with TMV, a few newly expanding leaves will, 3 to 4 days later as a result of systemic infection, develop a visual symptom called vein-clearing. This symptom signifies a synchronous production of TMV in the mesophyll cells of the leaf and is distinct from the mosaic symptom appearing on leaves that expand thereafter. Vein-cleared leaves were obtained by the standardized procedure of Nilsson-Tillgren et al. (1989). All experiments were carried out on material from a single batch of plants sown and grown together, selected for similar size and appearance, and infected simultaneously. (b) Labelling Leaves were cut into strips and labelled with [5-3H]uridine Amersham) for 30 or 120 min by the method of Nilsson-Tillgren
(60 &X/ml, 5 Ci/mmol, (1969,197O).
(c) RNA eztmction RNA was extracted at 0°C by the phenol/detergent method described by KiellandBrandt & Nilsson-Tillgren (19733). Purification was as described in the same paper with the exception that DNAaae digestion was carried out for 1 h in 0.15 M-NaCl, 0.01 MM@&, 0.02 m-Tris*HCl (pH 7.2) at 37°C. (d) l%actionation of RNA The RNA was chromatographed on a cellulose column by the method of Franklin (1966) as modified by Bishop & Koch (1969). The RNA eluting in buffer containing 15% ethanol
BIOSYNTHESIS
OF TOBACCO
MOSAIC
VIRUS
491
was collected as the single-stranded RNA fraction and precipitated with 2 vol. ethanol after addition of 0.1 vol. 3 ~sodium acetate, pH 5. It was dissolved in SSC buffer (O-15 MNaCl, 0.015 ~-sodium citrate, pH 7) and istored frozen at -20°C. The RNA eluting in ethanol-free buffer was rechromatographed twice, discarding material eluting in buffer containing 16% ethanol. After addition of E.&mkhk coli RNA to 50 pg/ml, the RNA wss precipitated with ethanol as described above. After dissolution of the RNA in SSC, the RI was precipitated (together with most of the E. COGRNA) by the addition of NaCl to 1.5 M according to Bishop & Koch (1967). The RI was chromatographed once more and the RI and the RF (the supernatant in 1.5 M-NaCl) were precipitated with ethanol as described above, dissolved in SSC, and stored frozen at - 30%.
(e) Non-radioactive RP Non-radioactive RF was prepared analogously by two cycles of chromatography. Its content of minus strands was determined according to Kielland-Brandt & NilssonTillgren (1973b). (f) TMV RNA The preparation of tritiated TMV RNA hss been described previously (KiellandBrandt & Nilsson-Tillgren, 1973a). Non-radioactive TMV RNA was prepared analogously from non-labelled leaves. (g) Melting and annealing Melting (strand separation) and annealing (strand association) were performed in 4546 formamide in SSC (0.3 ml) covered with paraffi oil (Kielland-Brandt & Nilsson-Tillgren, 1973a). Melting was achieved by heating to 91°C for 3 min. Annealing wss performed at 57°C for 5, 40, or 180 min as specified. (h) Radiochemical methods Determination of the RNAase-resistant radioactivity and the total radioactivity in RNA was according to Kielland-Brandt & Nilsson-Tillgren (19730). However, the RNAase treatment was reduced to 30 min. The background (filter with no tritium) was 23 cts/min. (i) Determinatkn of RNA The concentration of the purified RNA was determined spectrophotometrically dilution in SSC taking its specific absorbancy at 260 nm ss 22.2 cm2mg-1.
after
(j ) Isotope d&h&n amay for determindion of plus strand.3 and radioactivity in plus strands in the single-stranded RNA fradona Five portions, each constituting 1140th of the radioactive sample, were melted and annealed with non-radioactive RF (0.147 fig minus strands per tube). To 4 of the tubes, different known amounts of non-radioactive TMV RNA were added prior to melting. After annealing for 180 min the RNAase-resistant radioactivity W&Bdetermined. Furthermore, two tubes were set up, each containing only one portion of the sample; one was subjected to melting and annealing whereas the RNAsse resistance of the other w&s determined without melting and without annesling. Since a large excess of plus strands was present in all tubes, the minus strands annealed completely (Kielland-Brandt t Nilsson-Tillgren, 1973a). Thus, for the 5 tubes containing non-radioactive RF the following equation can be set up: R* = p*
(B + M)
P+
A
+‘*’
where R* is RNAsse-resistant radioactivity determined after melting and annealing; P+ is the radioactivity in plus strands; B is the amount of added non-radioactive minus strands; 1cf is the amount of minus strands in the single-stranded RNA fraction (residual RF and RI);
492
31. C. KIELLAND-BRANDYP is the amount
of plus strands in the single-stranded RNA fract,ion; A is the amount of added non-radioactive plus strands ; C* is RNAaae-resistant radioactivity not due to annealed plus strands. It includes radioactivity in remaining acid-precipitable oligonucleotides and radioactivity in the minus strands of the residual amounts of RF and RI. In each series of 5 tubes B was kept constant, A was varied, and the variable Iz* w&s measured. To find P* and P it was necessary to consider C* and M. The calculation was performed as follows. (i) A preliminary estimate of P*/P was calculated from R*, B, and D* by the approximation R* = P*B/P + D*, on the tube having received non-radioactive RF but no TMV RNA. D* is the RNAase resistance of a sample determined in 0.05 x SSC; it constitutes the majority of C*. (ii) In the tube in which melting and annealing wero carried out on a sample with no additions, the RNAase-resistant radioactivity should bc: R,*
= M(l’*/P)
(iii) The RNAase-resistant radioactivity melting and annealing, can be written as Ho*
+ C*.
that
= MS0
(2)
was determined + c*,
directly,
i.o. without (3)
where S, is defined by setting MS0 equal to the RNAase-resistant radioactivity in the plus strands of the residual RF and RI present in the single-stranded fraction. AS’,,was estimated by taking a weighted mean of the corresponding value found for the RF fraction and that for the RI fraction (radioactivity in base-paired parts of plus strands per pg of minus strands, Table 5, line (D)). This is correct only if M does not include free minus strands. As found by Siegel et al. (1973) and in the present study, free minus strands are absent or low in amount. (iv) From equations (v) Equation
(2) and (3) C* and 41 were found.
(1) can also be formulated A(R*
-
C*) = P*(B
as -I- M)
-
P(R*
- C*).
Accordingly, A(R* - C*) ww;“, plotted against R * - C*. Plotting and linear regression analysis were carried out using a Hewlett-Packard Calculator 91OOA and plotter 9125B with program no. 70818. P* is found from the intercept with the ordinate by dividing by B + M. P is the slope with opposite sign. With this better estimate of P*/P, steps (iv) and (v) were repeated. This single iteration changed the results insignificantly. The or (A-l, ((R* - C*)A)-l) also offer methods for pairs of variables (A,(R* - C*)-I) finding P and P* by linear plotting. They could have been used instead.
3. Results (a) Properties
of the extracted radioactive
RNA
Tobacco leaves were systemically infected with TMIV. The procedure used leads to a fairly synchronous virus synthesis in the cells of the leaf parts showing the veinclearing symptom (Nilsson-Tillgren et aE., 1969). At three different stages of virus production these leaf parts were labelled with [3H]uridine and the RNA was extracted. At two to eight hours after vein-clearing, virus has just begun to accumulate in the cells. 26 to 32 hours after vein-clearing represents the stage where virus synthesis is at its maximum. At 74 to 80 hours the virus synthesis has again decreased (Nilsson-Tillgren et al., 1969). Some characteristics of the purified RNA are given in
BIOSYNTHESIS
OF TOBACCO TABLE
MOSAIC
493
VIRUS
1
Tritiated RNA from infected leaves Stage of virus production (time after vein-clearing) Labelling period (min)
(h)
Amount of RNA extracted (mg) Specific radioactivity (cts/min per pg) RNA&se resistance in SSC buffer (%)
2-8 30
26-32 30
7680 30
2-8 120
26-32 120
74-80 120
5.6 120 3.1
6.1 163 3.9
5.7 187 3.2
6.5 1049 2.0
7.7 994 2.1
6.4 1226 2.0
Table 1. As expected (Nilsson-Tillgren, 1969), the RNAase resistance is lower with the long than with the short labelling time. (b) Fractiondio?L of the RNA Each of the six RNA preparations presented in Table 1 was separated into a singlestranded fraction, an RF fraction, and an RI fraction as described in Materials and Methods. The recoveries of RF, RI and single-stranded RNA from the purified loaf RNA through the total fractionation are given in Table 2 ; also the RNAase resistance of the fractions. Double-stranded RNA is resistant to RNAase when tested in SSC buffer but sensitive in 0.05 x SSC. Single-stranded RNA is sensitive at both salt concentrations. It can be seen that the amount of double-stranded RNA in the singlestranded fraction was small. The recoveries given for RF and RI are based on the TABLE
2
Properties of the RNA fractions Stage of virus production (time after vein-clearing) L&belling period (min)
(h)
2-8 30
26-32 30
74-80 30
2-8 120
26-32 120
74-80 120
Single-stranded RNA fraction Recovery of RNA&se-sensitive radioactivity from the purified leaf RNA (%I RNAase resistance : in SSC (%) in 0.06 X SSC (%)
94 0.39 0.11
97 0.24 0.07
99 0.22 0.14
99 0.25 0.11
100 0.29 0.16
99 0.24 0.16
RI fraction Recovery of RI from the purified leaf RNA (bred on RNA=-resistrtnt radioactivity) (Oh) RNAase resistance : in SSC (%) in 0.05 X SSC (Oh)
64.3 54.0 0.1
49.0 44.0 0.2
37.3 27.1 0.0
45.4 29.4 0.0
60.5 29.6 0.0
46.8 23.2 0.0
RF fraction Recovery of RF from the purified lesf RNA (based on RNAase-resistant radioectivity) (Oh) RNA&we resistance : in SSC (%) in 0.06 x SSC (%)
61.6 84.7 0.0
60.3 93.6 0.3
37.3 83.0 0.1
52.1 81.4 0.1
66.7 75.7 0.1
54.1 76.7 0.1
494
M. C. KIELLAND-BRANDY?
assumption that they have the same recovery when they are chromatographed together. As will be seen below (Table 5, line (G)) the RF to RI ratios calculated from these recoveries (and the minus-strand content of the fractions) are similar to those found earlier (Kielland-Brandt & Nilsson-Tillgren, 19733). (c) Virus-specific RNA ila the RF and RI )wGo~zs The RNAase resistances given in Table 2 indicate that the RF fractions are not radiochemically pure, since TMV RF has been prepared earlier that was more resistant to RNAase (Nilsson-Tillgren, 1970; Kielland-Brandt $ Nilsson-Tillgren, 1973a). In order to find out how much of the RNAase-sensitive radioactivity in the RF and RI fractions (Table 2) resided in virus-specific RNA, portions of the fractions were annealed together with melted non-radioactive RF (Table 3). Since the difference between the two columns is insignificant, it appears that the RF and RI fract,ions did not contain detectable amounts of labelled virus-specific single-stranded RNA. It was ascertained that radioactivity in single-stranded RNA would anneal in this particular experiment by annealing, in parallel, tritiated TMV RNA with the melted non-radioactive RF. Thus little-probably less than 20a/o-of the radioactivity in the RI molecules seems to be present in the single-stranded regions. The RNAase-sensitive material in the RF and RI fractions must be due to host RNA not removed by the fractionation. TABLE
3
Virus-specijc RNA in the RF and RI fractions Stage of virus production (time after vein-clearing) (h) 2-8 2-8 26-32 26-32 74430 74-80 2-8 2-8 2632 26-32 74-80 74-80
Labelling period WN 30 30 30 30 30 30 120 120 120 120 120 120
RNAase-resistent Fraction
RI RF RI RF RI RF RI RF RI RF RI RF
In sample
287 509 504 880 232 686 1785 2478 2691 3977 1913 3319
radioactivity (cts/min) After annealing with melted, non-radioactive RF? 285 621 611 847 242 662 --t 2497 2811 4023 1886 3242
t Non-radioactive RF (1.28 pg minus strands per ml) was melted and quickly oooled in a - 16’C bath. 100 4 of this melted RF was immediately added to a portion of each fraotion, and the mixtures were subjected to annealing conditions for 40 min before determination of RNAaseresistant radioactivity. $ Not determined.
(d) Radioactivity in plus and minus strands of RF and RI Samples of the six RF fractions and the six RI fractions were melted and annealed with (i) different amounts of non-radioactive TMV RNA, and (ii) different amounts of tritiated TMV RNA. The first procedure follows the specific isotope dilution assay
radioactivkyt
(time
neither
(iii)
melting
127.7
13.3
141.0
“77.0
14.5 124 14.4 12.6 24.1
2-R 30 RI
“40.5
13.5 244.3
12.2
368.3 ‘86,5
250.3
13.5 46.5
16-4
11.7 9.2
2632 30 RI
“54.0
14.8 13.6 13.3 13.7 33.8
“- 8 30 RF
423.3
13.3
436.6
403.2
11.7 10.2 14.0 21.6 70.6
26-32 30 RF
and annealing
163.5
115.3
6.3
121.6
277.7
5.8
283.5
294.6
6.7 7.9 35.7
4.9 5.0
RF
RI
7.8 6.4 3.9 7.7 12.2
7680 30
7P80 30
776.0
126.4
902.4
387.6
‘00.5
119.9
134.7 127.5 128.2
RI
2-8 120 RI
RF
396.9
1051.7
129.6
1181.3
1258.2
75.8
1334.0
649.6
74.4 73.0 83.4 87.4 288.7
26-32 120
2-8 120
127.7 131.3 133.7 131.6 220.5
of the RF and RI fractions
4
1864.0
72.0
1936.0
688.2
88.5 76.3 408.0
69.1
76.2
RF
26-32 1’0
874.0
51.5
9PB.Q
421.0
1511.5
51.4
1562~9
510.7
51.9 48.1 58.1 60.8 279.4
RF
RI
48.6 62.1 53.9 55.3 146.2
74-W 120 74-80 120
t Cts/min per sample. Each sample was 1/30th of the fraction. The values have been correct,ed by subtracting the RNAase-resistant radioactivity of an equal sample determined in 0.05 x SSC. It was between 0 and 2 cts/min. $ Bnnealing for 5 min. The rest of the Table contains values determined after 180 min annealing. $ Four different amounts (50, 10.3, 3.5 and 1.2 pg) of tritiated TMV RNA of a specific radioaotivity of 7613 cts/min per pg. The RNAase-resistant radioactivity was corrected by subtracting O.OZ”h of the radioactivity in the tritiated TMV RNA, since that was the RNAese resistance of this RSA. Even t,he lowest amount of added tritiated TMV RNA represents a large excess of plus strands and therefore the four values should be equal. The average in given.
of plus
nor annealing
melting and annealing TMV [3H]RNA $
Estimated radioactivityt in minus strands in base-paired parts strands
after with
(ii)
11.1 /%t 0.37 fig
370 /& 11.1 N
(i) after melting and annealing with non-radioactive TMV RNS, 370 Pg
RNAase-resistant
Stage of virus production after vein-clearing) (h) Labelling period (min) Fra&ion
Jleltiqy
TABLE
496
hf. C. RIELLAND-RRANDT
of Weissmann et al. (1964) and the second the isotope dilution assay of KiellandBrandt t Nilsson-Tillgren (1973b). The RNAase-resistant radioactivity determined after melting and annealing is given in Table 4. (i) Melting and annealing with non-radioactive tobacco mosaic viru.s RNA At the two highest concentrations of plus strands, 370 pg and 11.1 pg per tube of 0.3 ml (Table 4), annealing should be practically complete after the first five minutes, as the rate constant is about 25 mg-lmin-lml (Kielland-Brandt t Nilsson-Tillgren, 1973a). 180 minutes of annealing give similar or slightly higher values, and not significantly lower values than an annealing period of five minutes. Therefore the decrease in radioactivity conferred by the addition of TMY RNA cannot be due to nuclease contamination but is a real competition phenomenon. At both concentrations practically the same RNAase-resistant radioactivity was obtained, showing that the non-radioactive plus strands competed all radioactive plus strands out. When 0.37 rg non-radioactive plus strands is added, the annealing should also be practically complete after the 180 minutes. This amount appeared to be insufficient to compete all radioactive plus strands out. To get the best estimate of the amount of radioactivity in minus strands, the RNAase-resistant radioactivity was plotted against the reciprocal of the amount of added non-radioactive plus strands. By extrapolation to the ordinate we obtained the radioactivity in minus strands given in Table 4. Samples of the fractions were also melted and annealed together with 0.037 tug non-radioactive TMV RNA and with no TMV RNA. Still more RNAaseresistant radioactivity than with the addition of 0.37 pg TMV RNA was the result (data not shown). The radioactivity in base-paired parts of plus strands (Table 4) is found by subtracting the radioactivity in minus strands from the RNAase-resistant radioactivity of an untreated portion of the fraction. This gives an error for the RI fraction if parts of the minus strands are non-base-paired. However, the error will be small, since so little of the radioactivity is in minus strands, From the data in Table 4 it was possible to estimate the total radioactivity in plus strands per sample (cf. formula (6) of Weissmann et al., 1964). It was found that the total radioactivity in plus strands did not differ significantly from the radioactivity in base-paired regions of plus strands. This result is in accordance with the data in Table 3. (ii) Melting and annealing with trztiated tobacco mosaic virus RNA The RNAase-resistant radioactivity after melting and annealing with tritiated TMV RNA is given in Table 4. After subtraction of the radioactivity in minus strands the value was divided by the specific radioactivity of the added tritiated TMV RNA, whereby the amount of minus strands per sample was obtained (Table 5, line (A)). From these values and additional data of Table 4, the specific radioactivity of minus strands, as well as the radioactivity in plus strands per /*g of minus strands, was calculated. The results are given in Table 5. It can be seen that the specific radioactivity of minus strands in each RI fraction is almost the same as in the corresponding RF fraction (line (B)). The specific radioactivity of minus strands is threeto fourfold that of the total leaf RNA at the earliest stage of virus production, but later it is about the same as that of the total leaf RNA (line (C-)). The radioactivity in plus strands relative to the amount of minus strands is somewhat, larger in RF
9.6
(E) Radioactivity in base-paired parts of plus strands relative to that in minus strands 0.60 0.79 250
(G) Amount of minus st~randa in RF divided by that in RI
(H) Cts/min in minus strand3 per mg of total RNA
pg of minus strands per mg of total RSAk
3778
(D) Cts/min in base-paired parts of plus strands per rg of minus strands:
(F)
3.3
(C) Specific radioactivity of minus st,rands relative to that of the tot,el RNA
30.2
33*8
17.8
7964
3.7
447
“-8 30 RF
Z-8 30 RI
394
of minus strands
of minus st.rands per sample
(time after
(B) Specific radioactivity (cts/min per pg)
(A) Nanograms
Stage of virus production vein-clearing) (h) L&belling period (mm) Fraction
20.0
5154
1.6
257
47.4
26-32 30 RI
230
0.89
0.90
31.8
8156
1.6
256
51.9
26-32 30 RF
.J nm/ T1.t.u of nai~,w .strand.s and radioactivity
(Oaq
170
47.9
7213
0.8
151
38.5
7P80 30 RF
(1.97)
18.3
(5883)
(1.7)
(321)
(19*6)
74s”so 30 R.1
6.X
0.89
0.67
8.1
29,540
3.5
3638
35.6
2-8 120 RF
in RF ad
2439
22,360
3.5
3643
34*7
‘-8 120 RI
in plus and rninu.s strands
TABLE 6
l&6
0.9
878
82,O
26-32 120 RF
91G
O.Y9
5.9
72.i30
0.9s
16,470
1.0
992
76.4
26-32 120 RI
RI
17,o
17,770
0.9
1047
49.2
74-80 120 RI
960
1.07
1.02
29.4
24,740
0.7
841
61.1
7680 120 RF
498
M. C. KIELLAND-BRANDT
than in RI; no change with stage of virus production is evident (line (D)). As a consequence, the ratio of label in plus strands to label in minus strands increases with time after infection and is greater in RF than in the corresponding RI (line (E)). From the recoveries during fractionation (Table 2) and the data of line (A) (Table 5), the amount of minus strands relative to the total amount of leaf RNA has been calculated (line (F)). Also the ratio of RF to RI is given (line (G)) as well as the radioactivity incorporated into minus strands divided by the amount of leaf RNA (line (H)). The RI values for the 30-minutes labelling at 74 to 80 hours after vein-clearing fall clearly outside the rest of the data: (i) in contrast to the other five labellings, the specific radioactivity of minus strands in RI is twice that in RF, though this is possibly not significant because of the low number of counts. (ii) The amount of minus strands is somewhat lower than in the corresponding 120-minutes labelling and lower than found by Kielland-Brandt t Nilsson-Tillgren (19733). (iii) The ratio of RF to RI is twice that found for the other labellings and that found by KiellandBrandt & Nilsson-Tillgren (19733). All three discrepancies are eliminated by assuming that the amount of minus strands in the RI fraction has been underestimated by a factor of two, because of some error made in the experiment. This possibility is indicated by the brackets in the Table. Accordingly, disregarding these values, the amount of minus strands was O*1o/oof the total RNA at the late stages and a little lower at two to eight hours after vein-clearing (line (F)). The ratio of RF to RI was around one at all three stages (line (G)). Line (H) indicates a preferential Rynthesis of minus strands at two to eight hours after vein-clearing. (e) Analysis of the single-stranded RNA fractions The single-stranded fractions were analysed for their content of plus strands and the radioactivity in the plus strands by the isotope dilution assay described in
50 R*-C*(cls/min)
FIG. 1. Determination of plus strands and radioactivity in plus strands in the single-stranded fraotions after 120 minutes of labelling. The isotope dilution assay used is desoribed in Materials and Methods. Abscissa: annealed radioaotivity in plus strands (R* - C*). Ordinate: amount of added non-rsdioactive TMV RNA times annealed radioaotivity in plus strands (A(R* - C*)). The amount of plus strands per sample is found 88 the slope with opposite sign. The radioactivity in plus strands per sample is found by dividing the intercept with the ordinate by the amount of minus strands per tube. --O-O--, --e-m-, 26 to 32 h after vein-clearing; -A-A-, 74 to 80 h 2 to 8 h after vein-clearing; after vein-clearing (the skew point is not included in the linear regression). The baokground, C*, was 246, 261, and 273 ots/min, respeotively. The correlation coefficient was -0.991, -O*QSS and -0.999, respectively.
BIOSYNTHESIS
OF
TOBACCO TABLE
MOSAIC
499
VIRUS
6
Xingle-stranded RNA fra&imLs, I20 minutes lube&g Stage of virus production
(time after vein-clearing)
(h)
Retio of plus strands to total RNA Retio of radioclctivity in plus strands to thst in the totd RNA Specific radioactivity of plus strands (cts/min per pg)
2-a
26-32
74-80
0.023
0.098
0.213
0.23 10,200
0.39 3960
0.27 1640
Materials and Methods. The data are given in Figure 1 and the results in Table 6. For the 30-minutes labelling the background of radioactivity in oligonucleotides p*, see Materials and Methods) was too large to allow any reasonable calculations, so only data after 120 minutes of labelling have been used. Table 6 shows that a massive plus strand synthesis was taking place during the three-day period investigated. From the data of Tables 5 and 6 were calculated the radioactivity in plus strands per pg of minus strands, the radioactivity in minus strands per pg of plus strands, and the ratio of plus to minus strands (Table 7). The radioactivity incorporated into plus strands per pg of minus strands was about the same at the three stages of virus production, whereas that incorporated into minus strands per pg of plus strands decreased more than 20 times. The ratio of plus to minus strands was increasing with time after infection. TABLE
7
Radioactivity in plus and minus strands after 120 minutes labelding Stage of virus production
(time after vein-oloaring)
(h)
Cts/min incorpormed into plus strands per pg of minus strands Cts/min incorporated into minus &rends per pg of plus strands Ratio of plus to minus &ends
2-8
26-32
74-80
348,000
394,000
321,000
107 34
9.4 100
4.6 208
By subjecting samples of the single-stranded fractions to annealing conditions together with tritiated TMY RNA without prior melting, no free (single-stranded) minus strands could be detected (data not shown). However, apart from the samples from two to eight hours after vein-clearing, this experiment was not very sensitive because of the presence of a large amount of plus strands with a low specific radioactivity.
4. Discussion (a) The jractionation Tables 2 and 3 show that the RI fractions contain a great deal of-and the RF fractions some-radioactive host RNA. The fourth chromatographic run of RI only changed the RNAase-resistant radioactivity a little (data not shown). In
500
M. C. KIELLAND-BRANDY-
separating RNA extracted from Sendai virus-infected cells, Portner & Kingsbury (1972) experienced similar difficulties with cellulose columns. The level of TMV sequences and its complementary sequences in RNA from uninfected plants is so low (Kielland-Brandt & Nilsson-Tillgren, 19733) that the incompleteness of the fractionation is negligible in the present context. The single-stranded fractions contained small amounts of RF and RI, which could probably have been removed by repeated chromatography. It was preferred to determine their amount and correct for them. (b) Amounts of plus arm! minus strands The data on amounts of minus strands and their distribution between RF and RI (Table 5) are in accordance with earlier determinations (Kielland-Brandt & NilssonTillgren, 19733). Determination of plus strands (Table 6) gave two to three times higher values than were obtained earlier, but the kinetics of their appearance confirm our observation that the ratio of plus strands to total RNA is steadily increasing whereas the ratio of minus strands to total RNTA soon reaches a constant (and low) level. The ratio of plus to minus strands (Table 7) corresponds well to that found in leaf homogenates (Nilsson-Tillgren et al., 1974) from plants grown during the same months. The differences in plus-strand content may therefore be due to small variations in the growth conditions. Kielland-Brandt & Nilsson-Tillgren (19736) found relatively large differences in plus-strand content when different batches of plants were analysed. The present experiments do not distinguish molecules of different size. Thus the TMV-induced low molecular weight RNA (Jackson et al., 1972) will simply be measured as plus-strand material (Siegel et al., 1973). However, the low molecular weight RNA represents only a minor part of the plus-strand material (Jackson et al., 1972). Most, if not all, minus strands were present in the double-stranded forms. This is in accordance with the results of Siegel et al. (1973) who did not find any free minus strands in an electrophoretic analysis. The majority of plus strands was in the single-stranded fraction. It was not possible to measure the amount of plus strands in the RI and RF fractions directly but the data on non-base-paired radioactivity in Table 3 indicate that it amounts to no more than a few times the amount of minus strands. (c) Asymmetry of lubdling of the double-stranded fornzs On the basis of annealing experiments Siegel et al. (1973) concluded that after two hours labelling the RF contains about equal radioactivity in plus and minus strands, As they point out, this is an unexpected finding. However, it was not ascertained that in the annealing with non-radioactive TMV RNA all radioactive plus strands were competed out; neither was it shown that the annealing with non-radioactive double-stranded RNA was complete. Earlier analyses of Nilsson-Tillgren revealed a pronounced asymmetry in the labelling of the double-stranded forms (cf. Nilsson-Tillgren, 1970). In the present study this asymmetry of labelling has been confirmed. Even after two hours of labelling the ratio of radioactivity in the plus strands to that in the’minus strands was between 6 and 29 depending on the stage of infection.
BIOSYNTHESIS
OF TOBACCO
MOSAIC
VIRUS
601
(d) Role of the double-stranded form It is well established that RF and RI have a precursor role for TMV RNA (NilssonTillgren, 1969,197O; Jackson et al., 1971,1972). The fact that the plus-strand material in RF and RI has a higher specific radioactivity than has the single-stranded TMV RNA (Tables 5 and 6) gives strong support to this notion. It is not known whether TMV RF and TMV RI are double-helical in viva. (e) Eficiency
of labelli’ng
The capacity of the cells for uridine uptake and the sizes of their nucleic acid precursor pools need not be the same at different stages after infection. Therefore, deductions from radioactivity to rate of synthesis in the comparison of different stages require a measure for the efficiency of labelling. The best measure in the present experiments is the specific radioactivity of the plus strands in RF. It should be equal to the specific radioactivity of the nucleic acid precursor pool shortly before termination of the labelling period. The specific radioactivity of plus strands in RF did not vary much with the stage of virus product’ion (Table 5, line (D)). (f) Rate of synthesis of plus strands In Table 7 it is seen that the radioactivity incorporated into plus strands per pg of minus strands was about the same at the three stages investigated. Since the efficiency of labelling was also about equal, the number of plus strands synthesized per minus strand per unit of time should be about the same. By dividing the radioactivity in plus strands per pg of minus strands by the specific radioactivity of plus strands in RF one obtains a minimum estimate of plus-strand initiations at a given minus strand during the two hours. It is found t,hat the average time between successive initiations at a given minus strand was maximally, ten minutes at 2 to 8 hours, seven minutes at 26 to 32 hours, and nine minutes at 74 to 80 hours after veinclearing. For other RNA viruses higher, as well as a lower, frequency of initiation has been indicated. Thus, since a poliovirus plus strand is completed in about one minute (Girard et al., 1965) and poliovirus RI contains several nascent plus strands (Baltimore, 1968), the average time interval between successive plus-strand initiations at a given minus strand should be less than one minute. Turnip yellows mosaic virus plus strands, on the other hand, have been proposed to be synthesized with the rate of one strand per hour at each site of synthesis in the cell (Ushiyama & Matthews, 1970). (g) Rake of synthesis of minus strands
When minus-strand synthesis is measured relative to the total amount of RNA or to total RNA synthesis, for the 120-minutes labelling it is about twice as high shortly after vein-clearing than at the two later stages investigated (Table 5, line (H)). For the 30-minutes labelling it is a little different, but with the low number of counts t,his is probably not significant. The specific radioactivity of the minus strands was, at the early stage, three- to fourfold that of the total RNA but at the later stages about the same as that of the total RNA. From these observations two conclusions can be drawn. First, there is a preferential synthesis of minus strands at or shortly after vein-clearing. Even on a per leaf basis the synthesis of minus strands must be higher at t’wo to eight hours after vein-clearing
602
M. C. KIELLAND-BHANDT
than at the two later stages. Second, the minus strands must be rather stable, at least at the later stages of virus production. This is deduced from the fact that at these stages the minus strands constitute a constant part of the total RNA and have about the same specific radioactivity as the total RNA. Therefore the relative rate of breakdown of minus strands can not be greater than the relative rate of breakdown of the total RNA. When minus-strand synthesis is measured relative to the amount of its template, plus strand (Table 7), a pronounced decrease with time is observed. The removal of plus strands by incorporation into virions is probably a major reason for this, but other mechanisms may aot as well. (h) Functions
of RF and RI
For the distinction between RF and RI their different solubility in 1.6 M-NaCI has been used in the present study. Nilsson-Tillgren (1970) has previously characterized in this laboratory TMV RF and RI prepared with the methods used here by sedimentation analysis in sucrose gradients, and demonstrated that precipitation with NaCl separates the double-helical RNA molecules with complete base-pairing from those containing single-stranded regions. As the specific radioactivity of minus strands is the same in RF and RI (Table 5, line (B)) and minus strands are not extensively broken down, a rapid interconversion of the two double-helical RNA forms during replication is indicated, in contrast to the situation in phage MS2 or poliovirus. Radioactivity in base-paired parts of plus strands per pg of minus strands was consistently higher in RF than in RI (Table 5, line (D)). Furthermore, no significant radioactivity was found in single-stranded viral sequences in the RI fractions (Table 3). These two results point to the possibility that the single-stranded regions of RI are minus-strand regions rather than plus-strand regions. This possible difference between the structure of TMV RI and phage MS2 RI is under further investigation.
5. Conchdons Minus strands are synthesized at the highest rate at vein-clearing or shortly after. Thereafter, during the main accumulation of plus strands, the minus strands arc synthesized at a twofold lower rate. As shown earlier, the amount of minus strands remains at O*1o/oof the total RNA during this period of time. Breakdown of minus strands is small. Most if not all minus strands are present in completely (RF) or partially (RI) double-stranded structures after the extraction. RF and RI are probably interconverted in the process of plus-strand synthesis. The precursor role of RF and RI for TMV RNA has been confirmed.
I am indebted to Dr T. Nilsson-Tillgren for introducing me to the study of TMV BNA replication and for his steady help and advice. Further, I wish to thank Professor D. von Wettstein for support and encouragement and Miss J. Verhein Hansen for skilful technical assistance. Financial support was provided by grant no. GM10819 from the U.S. Public Health Service, National Institutes of Health and the Carlsberg Foundation to Professor D. von Wettstein.
BIOSYNTHESIS
OF TOBACCO
MOSAIC
VIRUS
503
REFERENCES Baltimore, D. (1968). J. Mol. Biol. 32, 359-368. Bishop, J. M. & Koch, G. (1967). J. Biol. Chem. 242, 17361743. Bishop, J. M. & Koch, G. (1969). Virology, 37, 521-534. Bishop, J. M. & Levintow, L. (1971). Progr. Med. Viral. 13, 1-82. Franklin, R. M. (1966). Proc. Nat. Acad. Sci., U.S.A. 55, 1504-1511. Girard, M. P., Baltimore, D. & Darnell, J. E. (1965). Fed. hoc. Fed. Amer. Sot. Erp.
Biol.
24, 379.
Jackson, A. O., Mitchell, D. M. & Siegel, A. (1971). Virology, 45, 182-191. Jackson, A. O., Zaitlin, M., Siegel, A. t Francki, R. I. B. (1972). V+roZogy, 48, 655-665. Kielland-Brandt, M. C. & Nilsson-Tillgren, T. (1973a). Mol. Gen. Genet. 121, 219-228. Kielland-Brandt, M. C. & Nilsson-Tillgren, T. (19735). Mol. Cen. Genet. 121, 229-238. Nilsson-Tillgren, T. (1969). Mol. Gen. Genet. 105, 191-202. Nilsson-Tillgren, T. (1970). Mol. Gen. Genet. 109, 246-256. Nilsson-Tillgren, T., Kolehmainen-SevBus, L. & von Wettstein, D. (1969). Mol. Gen. Genet. 104, 124141. Nilsson-Tillgren, T., Kielland-Brandt, M. C. & Bekke, B. (1974). 1cfoZ.Gen. Genet. 128, 157169. Portner, A. & Kingsbury, D. W. (1972). Virology, 47, 711-725. Siegel, A., Zaitlin, M. & Duda, C. T. (1973). ViroZogy, 53, 75-83. Ushiyama, R. & Matthews, R. E. F. (1970). V’iroZogy, 42, 293-303. Weissmann, C., Borst, P., Burdon, R. H., Billeter, M. A. & Ochoa, 8. (1964). Proc. Nat. Acad. Sci.,
1T.S.A. 51, 890-897.