Cell, Vol. 7, 567-573,

April

1976,

Copyright0

1976

by MIT

Preferential Synthesis of Viral Late RNA by Nuclei Isolated from SV40 Lytically Infected Cells Eli Gilboa and Haim Aviv Department of Biochemistry Weizmann Institute of Science Rehovot, Israel

Summary Nuclei from SV40-infected monkey cells were isolated late in lytic infection and their cell-free transcriptional activity was characterized. 3H-RNA synthesized in vitro was hybridized to excess quantities of separated SV40 DNA strands which were each covalently bound to Sepharose. It was found that 3-5% of the newly synthesized RNA is virus-specific and that the plus-strand DNA, coding for late RNA sequences, is transcribed at a rate about 15 times higher than that of the minus-strand DNA, which codes for early RNA sequences. This indicates that transcriptional control has a major role in determining the relative abundancy of early and late RNA classes in lytically infected cells. Introduction The synthesis and metabolism of SV40 virus-specific mRNA have many features in common with those of eucaryotic cellular mRNA (Weinberg, Warnaar, and Winocour, 1972; Jackson and Sudgen, 1972; Aloni, 1974; Lavi and Shatkin, 1975) and can therefore serve as a convenient model for studying the control of eucaryotic gene expression. Several specific eucaryotic mRNAs such as hemoglobin, ovalbumin, and immunoglobin mRNA are difficult to study because they are usually synthesized in quantities too small to be detected by presently available techniques. The SV40 system has several distinct advantages. First, SV40-specific RNA is synthesized in a relatively high proportion, 5-7% of the total RNA synthesized in lytically infected cells is viral RNA (Aloni, Winocour, and Sachs, 1968; Oda and Dulbecco, 1968). Second, viral DNA can be purified in sufficiently large quantities, and hybridization methods with very low levels of nonspecific hybridization are available, permitting the detection of low levels of viral RNA synthesis. Early RNA appears in the cytoplasm of infected cells before the onset of viral DNA synthesis and is transcribed from the minus-viral DNA strand. In lytically infected cells, viral RNA appears in the cytoplasm after the onset of viral DNA synthesis and is transcribed from the opposite DNA strand (the plus strand). Late RNA is 30-50 fold more abundant than early RNA (Aloni et al., 1968; Oda and Dulbecco, 1968; Khoury, Byrne, and Martin, 1972; Khoury and Martin, 1972; Lindstrom and Dulbecco, 1972;

Sambrook, Sharp, and Keller, 1972; Khoury et al., 1973a). In SV40 and polyoma viruses, only two transcriptional units have been identified, one for early and the other for late RNA. The 5’ termini of both early and late RNA are located near the origin of replication of the genome, and the two viral DNA strands are transcribed in opposite directions (Khoury et al., 1973a, 1973b; Sambrook et al., 1973; Weinberg, Ben-lshai, and Newbold, 1974; Kamen et al., 1974; Dhar et al., 1974). Aloni and other investigators (Aloni 1972, 1973; Aloni and Locker, 1973; Kamen et al., 1974; Khoury et al., 1975) have observed that self-complementary SV40 and polyoma RNA sequences which represent transcripts of the entire viral genome are present in nuclei of infected cells. In the cytoplasm, however, early and late viral RNA classes are not complementary to each other. Thus it was proposed that the viral genome is transcribed primarily in a symmetrical manner and that the RNA is then processed to remove complementary “anti-early” or “anti-late” sequences (Aloni, 1972, 1973). These studies, however, did not provide evidence regarding whether the accumulation of 30-50 fold more late RNA than early RNA in the cytoplasm of late lytically infected cells results solely from posttranscriptional processing of viral RNA, or whether there is preferential transcription of the plus-DNA strand over the minus-DNA strand. In this paper, we report that in nuclei isolated from SV40-infected cells late in lytic infection, the plus-viral DNA strand is preferentially transcribed, yielding approximately 15 fold greater amounts of late RNA sequences than the minus-DNA strand. Essentially similar results have been obtained recently by Laub and Aloni (1975). Results Characterization of Transcriptional Activity of Isolated Nuclei Reeder and Roeder (1972) have observed that the transcription pattern of rRNA genes in isolated nuclei (intact or lysed) is similar to that in whole cells. Transcription was no longer regulated, however, when chromatin was transcribed either with procaryotic or eucaryotic RNA polymerases (Reeder, 1973). We therefore decided to study first the synthesis of viral RNA in intact nuclei isolated from infected cells. This approach could be extended later to study the transcription of chromatin and to investigate the effect of associated components on the transcription process. Optimal conditions for transcriptional activity of nuclei isolated from BSC-1 monkey cells, 48 hr after

Cell 568

infection with SV40, were first determined. The labeled RNA synthesized was purified and then hybridized to separated SV40 DNA strands, each strand covalently bound to Sepharose (Gilboa, Prives, and Aviv, 1975). Total incorporation of 3H-UMP into RNA was higher with ammonium sulfate than with potassium chloride, at an optimal concentration of 0.15 M (Figure IA). The optimal concentration of MnC12 was 3 mM with a minor incorporation peak at 1 mM (Figure IB). The synthetic activity at 25°C proceeded linearly for 20 min and continued for at least I hr, but was slightly slower than at 32°C (or 37°C) (Figure 2). This is in contrast to the observation of Marzluff and his co-workers, using myeloma nuclei where incorporation at 25°C proceeded linearly for at least 60 min and was higher than at 37°C (Marzluff, Murphy, and Huang, 1973). 3 x 105 nuclei incorporated 1 pmole 3H-UMP in 20 min, and the activity was dependent upon the nuclei concentration (Figure 3). The total transcriptional activity showed the characteristics of DNA-dependent RNA synthesis. Omission of ATP, GTP, and CTP inhibited 3HUMP incorporation. The radioactive product was digested by RNAase and its synthesis was inhibited by actinomycin D (Table 1). The involvement of RNA polymerases I, II, and III in the synthesis of RNA was measured using a-amanitin, which does not inhibit polymerase I, but inhibits p.olymerase II and III (Zylber and Penman, 1971; Weinman and Roeder, 1974). a-Amanitin scarcely inhibited 3HUMP incorporation at low ionic strength, that is, when no ammonium sulfate was added. This is consistent with previous in vitro findings that at low ionic strength, the major synthetic activity is contributed by polymerase I, while at higher salt con, ‘,a

(A)

.

r

(6)

1. Ionic

Table 1. Characterization Nuclei from SV40-Infected

of 3H-RNA Synthetic BSC-1 Cells SH-UMP

Complete Omit

Nuclei

Omit ATP,

GTP,

+ RNAase

A

Activity

cm

Incorporated %

7160

100

220

3.1

20

0.3

CTP

680

+Actinomycin

D, 10 pg/ml

+ Actinomycin

D, 50 &ml

in Isolated

9.5

2300

32

630

8.7

1.6 x 10s nuclei were incubated in 0.05 ml of the standard reaction mixtures. 0 time background count of 800 cpm was subtracted. Pancreatic RNAase A (40 pg/ml, Worthington) was heated before use for 10 min at 80°C.

I

I

I

I

I

I

I

I

l-u

bl6 x

?J lOA

KCI

I

01 0.2 03 (NH412S04,KCI (Ml

Figure Nuclei

centrations, polymerases II and III become active (Reeder and Roeder, 1972). At a higher ammonium sulfate concentration (0.15 M), 70% of the synthetic activity was inhibited by 0.1-I pg/ml of a-amanitin, a concentration which inhibits RNA polymerase II; almost 100% of RNA synthesis was inhibited by 150 pg/ml of a-amanitin-a concentration at which RNA polymerase III is also inhibited (Figure 4). The biphasic pattern of inhibition by oi-amanitin is similar to that given in previous reports (Jackson and Sudgen, 1972; Weinman, Raskas, and Roeder, 1974). The involvement of RNA polymerase III in

Dependence

0.4

1234567 Mn+”

of XH-UMP

, Mg+’

(mt.4)

Incorporation

by Isolated

(A) Effect of ammonium sulfate and potassium chloride concentration on SH-UMP incorporation by isolated nuclei in vitro. Nuclei (2 x 105) were incubated for 20 min at 25°C in a standard reaction mixture of 0.05 ml. For conditions of the reaction see Experimental Procedures. (6) Effect of manganous chloride and magnesium chloride on ‘HUMP incorporation. 1.4 x 105 nuclei were incubated as described above.

TIME

(min.)

Figure 2. Effect of Incubation Temperature UMP Incorporation by Isolated Nuclei

on the Kinetics

of XH-

Nuclei, 2.7 x 106, were incubated in 1 ml of reaction mixture; quots of 0.02 ml were assayed at the indicated times.

ali-

Biosynthesis 569

of SV40

RNA

SV40 RNA synthesis could be excluded, since the synthesis of SV40 viral-specific sequences was completely inhibited by 5 pg/ml a-amanitin (not shown). This is in contrast to the situation in adenoinfected KB cells (Price and Penman, 1972; Weinman et al., 1974).

valently bound to Sepharose (see Experimental Procedures). Excess DNA hybridization conditions were chosen to detect the synthesis of all newly synthesized RNA molecules. The ratio of late to early RNA synthesized is in the range of lo-20 fold (Table 2). Of the RNA synthesized by the nuclei, 0.150.4% was early RNA. Nuclei prepared by lysis of cells with hypotonic buffer are as active in 3H-UMP incorporation as nuclei prepared by lysis with 0.5% Triton X-100, and no significant differences were observed in the synthesis of viral-specific early or late RNA. Once frozen, nuclei lose very little of their transcriptional activity; however, the synthesis of viral-specific RNA is somewhat reduced. The preferential transcription of the DNA strands is not changed by using nuclei prepared in different ways or by using frozen nuclei (Table 2). To ensure that the hybridization conditions chosen were indeed those of excess DNA, the hybridization was carried out with saturating

Rate of Early and Late Viral RNA Synthesis The rates of transcription of early and late viral DNA strands were measured by incubation of nuclei with labeled ribonucleotide triphosphates for a relatively short period of time (15 min). RNA was then isolated from the nuclei and hybridized to the separated strands of SV40 DNA, each of which had been coI

I

I

I

I

I 1

I

I

I

I

20t

-\ 10-4

NUMBER OF NUCLEI xIO-~

10-3

10-Z

10-l

a-Amanitin Figure 3. JH-UMP of Nuclei The standard Procedures).

Table

Incorporation assay

of 3H-RNA

was

used

Synthesized

(see

in Vitro

Experimental

Unfrozen

Detergent Frozen Detergent

Hypotonic

-I

IO’

102

(pg/ml)

Nuclei

3H-RNA

Hybridized

of JH-UMP

transcription

to Plus and Minus

Strands

incorporation assay

of SV40

was

by a-Amanitin used

(see

Experimental

DNA

to DNA Strands

(E)

Plus (L) %

Plus/Minus (Ratio)

4.1

13.6

cm

%

0.4

120

0.30

0.8

153

0.39

1447

7.2

18.5

1.6

149

0.38

1317

6.6

17.3

0.4

48

0.13

605

3.0

23.1

52

0.13

419

2.1

16.2

109

0.25

494

2.5

a.3

DNA Hypotonic

4. Inhibition

The standard Procedures).

by Isolated

Minus Nuclei

100

0.8 0.8

(,a)

I

of Concentration Figure

transcription

2. Hybridization

as a Function

I

wm

818

All the methods used-nuclei preparation, DNA strand separation, and coupling to Sepharose-are described in Experimental Procedures. The values of hybridization obtained by SH-RNA from mock-infected nuclei were in the range of 0.10% and were subtracted from the hybridization values of 3H-RNA from viral-infected nuclei. The input of 3H-RNA for hybridization to the plus strand was 2 x 104 cpm (obtained from 1.5 x 105 nuclei), while the input of 3H-RNA for hybridization to the minus strand was 4 x 104 cpm (obtained from 3 X IO5 nuclei). Since the UTP pool in the nuclei seemed negligible, it is assumed that the specific activity of the RNA synthesized is 3 x 107 cpm/m.

Cell 570

amounts of minus- and plus-DNA strands (Figure 5). The data indicate that the rate of late RNA synthesis is higher than that of early RNA synthesis. However, our hybridization technique has the following possible pitfall: the hybridization kinetics of immobilized DNA are more complicated and the rate of hybridization may be slower than in solution, conceivably leading to an error in the estimation of RNA sequences present in low concentration (Flavell, Borst, and Birfelder, 1974a; Flavell et al., 1974b; Spiegelman, Haber, and Halvorson, 1974). This arises because “late” RNA, present in a relatively high concentration in nuclei from lytically infected cells, may be complementary to “early” RNA, thus sequestering newly synthesized “early” RNA by forming a double-stranded RNA. If the formation of double-stranded early and late RNA in solution is kinetically favored over hybridization of early RNA to the minus-DNA strand which is cova-

0.4

0.8 DNA (pg/ml 1

1.6

Figure 5. Hybridization of SH-RNA Synthesized by Isolated Nuclei to Different Concentrations of Plus- and Minus-DNA Strands Details

Table

as in Table

3. Effect

2.

of Nuclear

RNA on the Hvbridization Minus

of SV40

3H-cRNA

lently bound to a solid phase (Flavell et al., 1974a, Spiegelman et al., 1974), this situation would have a greater effect on the detection of “early” RNA because it is present in much lower concentrations than “late” RNA. However, measurements of the effect of added nuclear RNA (nonlabeled) on the hybridization of 3H-cRNA to the separated viral DNA strands showed no such effect. If nuclear RNA, which has a higher concentration of late RNA, could form a double-stranded RNA with the anti-late fraction of cRNA, a reduction in the hybridization of cRNA to minus-strand DNA should be observed. Table 3 shows that addition of nonlabeled nuclear RNA did not affect the hybridization pattern of cRNA to DNA Sepharose. Another factor that could lead to the unequal hybridization of early and late RNA is the possibility that even in a 16 min incubation, early RNA is degraded more rapidly than late RNA. However, when nuclei were incubated for 5 min with labeled 3HUTP and then chased with cold UTP for another 60 min, the ratio of late to early RNA synthesized was not significantly affected by further incubation for another 60 min in the presence of unlabeled UTP (Table 4), thus excluding any effect of unequal degradation rates. That the separated DNA strands were not crosscontaminated was shown first by reannealing the separated DNA strands; less than 2% contamination was observed. However, since the low specific activity of the DNA did not permit more sensitive detection, we used the following more rigorous criterion. Labeled cRNA transcribed from the minus-DNA strand was hybridized to the separated DNA strands covalently bound to Sepharose. Only about 0.1 (1tO.03)% of the cRNA hybridized to the plus strand, while 33 (*2)% hybridized to the minusDNA strand under the given experimental conditions (in the presence of RNAase) (Figure 6). In addition, viral-specific late 3H-RNA was purified from the cytoplasm of late lytically infected cells by selective hybridization to plus-strand DNA Sepharose. 63% of this RNA (6950 cpm) hybridized to the plusstrand DNA, but only 0.5% to the minus-strand. Furthermore, hybridization of the newly synthesized

to Plus-

and Minus-DNA

Strands

Plus (L)

(E)

Plus/Minus (Ratio)

RNA Addition

cpm

%

cm

%

None

926

34.3

962

36.4

1 .I

945

35.0

974

36.1

1 .o

Nuclear

RNA

3H-cRNA was prepared as described in Experimental Procedures with a low concentration (2 x 10-S M) of 3H-UTP (49 Ci/mmole). Sonicated viral DNA served as template for the reaction, which yielded highly labeled cRNA (3 x 107 cpm/ug), which was rather symmetric, AH-cRNA (2700 cpm) was denatured and hybridized to plus- and minus-DNA strands in the presence and absence of nonlabeled nuclear RNA extracted from 3 x 105 nuclei.

Biosynthesis 571

of SV40

RNA

nuclear RNA to the minus-DNA strand could be prevented by competition with excess cRNA asymmetrically transcribed from the minus-DNA strand, but hybridization of RNA to the plus-DNA strand was not decreased by this cRNA (not shown).

DNA strand is higher than that of the early DNA strand. The presence of anti-early and anti-late viral RNA in nuclei from viral-infected cells (Aloni, 1972, 1973; Kamen et al., 1974; Khoury et al., 1975) indicates that either the termination signal of transcription is not efficient and read-through of late genes along with early genes is common, as suggested by Kamen et al. (1974), or that the complete genome is transcribed symmetrically and then processed in the nucleus to yield an asymmetric product, as suggested by Aloni (1972, 1973). The presence of symmetrical (double-stranded) RNA, however, is not related to the question of the differential transcription rates for early and late viral DNA strands, That these double-stranded RNA sequences do not appear in the cytoplasm implies that there is a posttranscriptional process either of degradation or perhaps selective transport which prevents these sequences from appearing in the cytoplasm. The accumulation of 30-50 fold more late RNA sequences than early ones in the cytoplasm of lytitally infected SV40 cells is a function of both the differential rates of DNA transcription and the differential degradation of the two types of RNA. From this report, it appears that the rate of late viral RNA synthesis is about 15 fold faster than early viral RNA synthesis, indicating that transcriptional control has a major role in the differential abundance of the two viral RNA classes. The relative contribution of differential degradation of viral RNA in the nucleus and the different half-lives of early and late viral RNA in the cytoplasm was not measured, but the above data indicated that the contribution would not be more than a factor of 3-5 in the determination of the amounts of late and early viral RNA accumulated. It is tempting to speculate on possible control mechanisms which may determine the differential transcription rates of early and late SV40 DNA strands. Among others, the following possibilities may be suggested. First, the template for early RNA transcription may be different from that for late RNA. Thus late RNA is transcribed from free-

Discussion We have shown in this series of experiments that the rates of transcription of “early” and “late” SV40 DNA strands are not identical. Late RNA sequences are synthesized about 15 fold faster than early RNA (Table 2, Figure 5), suggesting that the synthesis of SV40 viral RNA is controlled mainly by a transcriptional process as proposed earlier by Kamen et al. (1974). Laub and Aloni (1975), in their recent in vivo studies of the transcription pattern of early and late SV40 viral RNA, have shown that in this system also the transcription rate of the late viral I

I

I

I

I

0Minus (E)0 0 T 0

-

4

8

I2 Time

Figure 6. Hybridization DNA Strands

Kinetics

I6 (hours)

of 3H-cRNA

20

24

to Plus-

and

Minus-

IH-cRNA (2.5 x 104 cpm) was hybridized to 0.4 ug of plus- or minus-viral DNA strands. Conditions of hybridization are as described in Experimental Procedures.

Table

4. Effect

of Incubation

Time

and “Chase”

on the Transcription % 3H-RNA

Incubation (Min)

Minus

Time Chase

5 15 5

+

“H-RNA was synthesized, purified, in the oresence of XH-UTP (IO-5 0.1 mM, and incubation continued

of Earlv

Hvbridized

and Late Viral RNA bv Isolated

Nuclei

to DNA

(E)

Plus (L)

cw

%

cm

%

Plus/Minus Ratio

114

0.29

716

3.6

12.4

48

0.13

605

3.0

23.0

93

0.22

658

3.3

15.0

and hybridized as described in Experimental M) is indicated. In the chase experiment, for an additional 30 min.

Procedures and the legend to Table 2. The time of incubation nonradioactive UTP was added to a final concentration of

Cell 572

replicating viral DNA molecules, and early RNA is transcribed from integrated viral DNA. Although recent studies have shown quite clearly that freereplicating viral DNA molecules serve as a template for viral RNA transcription (Green and Brooks, 1975; Mousset and Gariglio, 1975), it is not clear yet whether the free-replicating DNA is a template for both early and late RNA. Alternatively, late and early viral RNA may be transcribed from two different types of free-replicating DNA molecules. One type of DNA molecule could serve as a template only for early RNA and would be initiated less frequently; the other type could serve as a template only for late RNA and would be transcribed with a higher efficiency. There could be several differences between these two types of transcriptional templates, one for early RNA and another for late RNA. For example, a “promoter” site replaced by host sequences (Lavi and Winocour, 1972) may change the efficiency of transcription differentially and thus enable the initiation of only early or only late RNA. Second, the early and late “operons” may be initiated with different frequencies because of a preferential affinity of RNA polymerase for the late operon. This can be a direct result of the nucleotide sequences of the initiation site itself, or may be due to positive control elements which may be either viral or cellular gene products which cause the late operon to have a higher affinity to RNA polymerase. Alternatively, negative control elements may render the early operon less available for initiation of transcription. An active cell-free transcription system such as we have described here would permit a more direct analysis of these possibilities. The relative simplicity of the SV40 genome, which contains only two transcriptional units, is an important advantage in studying the molecular aspects of transcriptional control of eucaryotic cells. Experimental

Procedures

Ceils and Viruses A line of African green monkey cells (BSC-1) and a plaque-purified stock of SV40 (strain 777) were provided by E. Winocour. Cells ware grown and infected with virus as described by Lavi and Winotour (1972). Preparation of Nuclei BSC-1 cells were infected with 50 PFU per cell. 48 hr later, the plates were washed twice with cold isotonic buffer [IO mM Tris-Cl (pH 7.5), 0.15 M NaCI]. The cells were then extracted and nuclei prepared by one of the following procedures: Hypotonic Extraction Plates were washed once with hypotonic buffer [I 0 mM Hepes-KOH (PH 7.5), 25 mM KCI, 3 mM MgCl2, 1 mM DTT, 0.1 mM EDTA]. 1 ml of buffer was applied to each plate. After IO min on ice, the cells were removed from the plates by means of a rubber policeman

and homogenized by 20-25 strokes in a Dounce homogenizer with a tight-fitting pestle. 0.1 vol of 50% glycerol was added per volume of lysate; the nuclei were pelleted for 5 min by centrifugation at 1000 x g and washed twice with hypotonic buffer containing 5% glycerol. Nuclei at a concentration of 2-4 x 107 ml were used either immediately or kept frozen in liquid nitrogen. Detergent Exfracfion Plates were washed with hypotonic buffer containing 5% glycerol, and 1 ml of buffer containing 0.5% of Triton X-100 was added per plate. After 5 min on ice, the cell lysates were harvested and homogenized by 5-10 strokes in a Dounce homogenizer and processed further as above. SV40 DNA Strand Separation S/40 DNA was prepared as described previously (Gilboa et al., 1975). DNA strands were separated essentially as described by Sambrook et al. (1972). Sonicated 3*P-SV40 DNA was hybridized with asymmetric complementary RNA and separated on hydroxylapatite. Each separated strand was then self-annealed and rechromatographed on hydroxylapatite; only the single-stranded DNA fraction was collected and used for covalent binding to Sepharose. Self-annealing of the DNA separated strands was less than 2%. Covalent Coupling of Separated Strands to The method used is described in detail by with the exception that CNBr was dissolved concentration of 2 g/ml and 0.1 g of CNBr Sepharose suspension (Sepharose H20, 1:l) Cuatrecasas, 1974).

Sepharose Gilboa et al. (1975), in acetonitrile at a was added per ml of (March, Marikh, and

Synthesis of RNA Complementary to SV40 DNA (cRNA) RNA was transcribed from 150 ug SV40 DNA by 100 units of E. coli RNA polymerase (Sigma) as described by Shih and Martin (1974), except that 2 mM ribonucleotide triphosphates were added; the incubation time was 3 hr. The final yield was 2.8 mg RNA. Under these conditions of synthesis(high XTP concentration), selfannealing of the RNA was not more than 4%. Synthesis of RNA by Isolated Nuclei Standard conditions were: 10 mM Hepes-KOH (pH 7.5), 0.15 M (NH&S04, 3 mM MnC12, 5 mM DTT, 0.1 mM EDTA, 0.1 mM ATP, GTP, CTP, and 5% glycerol. 2.5 UC of 3H-UTP (49 Ci/mmole, 13500 cpm/pmole) and 1-2 X 105 nuclei were added to each 0.05 ml reaction mixture. Incubation was carried out at 25°C for 20 min. The reaction was stopped by adding 0.2 ml of 0.2 M pyrophosphate, followed by cold 10% TCA. 10 min later, the acid-insoluble material was collected on nitrocellulose filters, which were washed with IO ml cold 5% TCA containing 20 mM pyrophosphate. In the preparation of 3H-labeled RNA for hybridization, the reaction mixture was scaled up IO-20 fold, and the concentration of 3H-UTP was doubled. About 5 x 105 cpm were incorporated per 1 ml reaction mixture, The RNA was deproteinized by hot phenol and treated with DNAase as described by Penman (1966). TCA-soluble material was removed by Sephadex G25, and the RNA was precipitated with ethanol and dissolved in a small volume of water. Hybridization of RNA to DNA Sepharose DNA Sepharose (0.4-1.6 ug DNA on 0.05-0.2 ml settled beads), 100 ug E. coli ribosomal RNA, and indicated amounts of 3H-RNA were incubated in 1 ml of hybridization buffer containing 20 mM Tris-Cl, 0.75 NaCI, 1 mM EDTA, 0.5% SDS, and 50% deionized formamide (Fluka puriss.) adjusted to pH 7.2, in a shaking water bath at 40°C for 22 hr. The suspension was washed with 10 ml of 2 x SSC (0.3 M NaCI, 0.03 Na citrate) and suspended in 2 ml of 2 x SSC. RNAase A [IO0 ug, preincubated for 10 min at 80°C in 0.01 M ammonium acetate (pH 5.0) and 0.1 M NaCI] was added; the suspension was shaken for 1 hr at room temperature, and

Biosynthesis 573

of SV40

RNA

washed with 3 ml of 2 x SSC containing 0.5% SDS and then with 3 ml of 0.01 Tris-Cl (pH 7.5). The hybridized RNA was eluted with 3 ml of 0.1 M NaOH and added to 5 ml lnstagel (Packard) for the radioactivity determinations.

We are grateful to Dr. C. Prives for many suggestions, discussions, and help during the course of this work. We thank Drs. Y. Aloni, U. 2. Littauer, M. Revel, and E. Winocour for helpful suggestions, and Ms. B. Yakobson and T. Koch for technical assistance. This research was supported by a grant from the National Cancer Institute and by the Israel National Academy of Sciences. October

20, 1975;

revised

January

16, 1976

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