Evidence for common nucleotide sequences in the RNA subunits comprising Rous sarcoma virus 70 S RNA

VIROLOGY

61, 287-291

(1974)

SHORT Evidence

for Common Comprising KRISTINA

of

Department

Microbiology

Nucleotide Sequences in the RNA Subunits Rous Sarcoma Virus 70 S RNA

QUADE, and

COMMUNICATIONS

Immunology,

R. E. SMITH, Duke University 27710 Accepted

May

AND

J. L. NICHOLS

Medical

Center,

Durham,

North

Carolina

3, 1974

Digestion of Rous sarcoma virus 32P-labeled high molecular-weight RNA with T, ribonuclease resulted in the release of approximately 30 unique large oligonucleotides which were resolved by electrophoresis and chromatography on thin layers of cellulose and DEAE-cellulose. Determination of the radioactivity present in each oligonucleotide of known chain length and in the remaining digestion products made it possible to estimate the total number of residues expected in a polynucleotide chain containing the sequences represented. The oligonucleotides are present in amounts greater than would be expected for a unique and nonrepetitive 70 S RNA molecule with a molecular weight of 1 x 10’. Many of the sequences must, therefore, be present in more than one of the 35 S RNA subunits comprising the 70 S RNA.

The genome of RNA tumor viruses has a sedimentation constant of 70 S corresponding to a molecular weight of about lo7 (2, 2). Denaturation of this RNA by heat or DMSO results in the release of three or four 35 S RNA chains (3-7), as well as several smaller RNA species (< 10 S) (3, 8-11). It is now clear that the 35 S RNA is not a degradation product of the viral genome but rather a precursor which becomes part of the 70 S RNA during virus maturation

thereby ruling out the possibility that each of the polynucleotide chains is completely different. The Prague strain of Rous sarcoma virus (subgroup C) was harvested from the medium of transformed cells grown in roller culture bottles (16). For radioactive labeling of the virus, the medium was as described previously (16) except that tryptose phosphate was omitted and [32P]orthophosphate was added to a final concentration of 200 kCi/ml. RNA was extracted from purified virus by phenol deproteinization and subjected to sucrose densitygradient centrifugation to separate the high molecular-weight RNA (70 S) from the smaller polynucleotide chains. The 35 S RNA recovered from this strain of Prague Rous sarcoma virus has been shown to consist exclusively of the a size class subunit. ’ The RNA was recovered by ethanol pre-

(12, 13).

Presently, it is not known whether the 35 S subunits have completely identical nucleotide sequences (polyploid model), or whether the genome consists of unique subunits, each potentially capable of specifying different polypeptide chains (haploid model) (14). Recently, evidence from molecular hybridization data has been presented to support a model in which each 35 S RNA subunit possesses a unique nucleotide sequence (15). The present report shows that extensive nucleotide sequences are repeated in the 35 S RNA subunits,

‘Stone, M. P., Smith, R. E., and Joklik, W. K. Cold Spring Harbor Svmp. &ant. Biol. (1974) (In press). 287

288

SHORT COMMUNICATIONS

/ / / /

/

2 26 o

8 27

& ba

,J

FIG 1. a. Autoradiograph of a thin-layer homochromatography plate after separation of a T, ribonuclease digest of 35 S RNA. b. Schematic drawing of the large oligonucleotides in Fig. la showing the numbering system employed to identify each component. 1, direction of electrophoresis in the first dimension; 2, direction of homochromatography in the second dimension.

SHORT

289

COMMUNICATIONS

termined by autoradiography. cipitation and digested with T, ribonuclease (17). The products of digestion were Figure la shows the oligonucleotide map separated in the first dimension by electroof a T, ribonuclease digest of 35 S RNA and Fig. lb shows a schematic drawing indicatphoresis at pH 3.5 in 7 M urea on cellulose ing the location of individual oligonucleoacetate strips and in the second dimension tides with the numbering system employed by homochromatography on thin-layer to identify each. The largest of the compoplates composed of a mixture of cellulose and DEAE-cellulose in a ratio of 7.5: 1 nents (spot 30) is poly (riboadenylic) acid were eluted (18). The developing solvent was a par- (19, 20). The oligonucleotides tially hydrolyzed solution of RNA in 7 M from the thin layers by the method of urea, pH 7.0. The position of the oligonuBrownlee and Sanger (18), and the chain cleotides on the thin-layer plates was de- length of each component was determined TABLE CHAIN

LENGTH

Oligonucleotide chain length

Average

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 chain

9 10 11 12 10 9 10 9 12 11 12 13 12 13 14 11 12 13 14 14 13 11 12 12 15 16 16 17 19 length

In some cases, certain components are, therefore, oligonucleotides are, on the be due to the fact that some acetate strip to the thin-layer 27,095, 491,061, and 448,556

ESTIMATES

1 OF RSV

Experiment

9,032 9,853

-

9,850 7,560 6,906 5,606 10,360 15,173 7,390 10,065

-

8,790

-

10,695 12,042 12,386 9,219 8,400 9,585

35s RNA 1

Experiment

8,626 6,710 5,778 5,840 3,951 11,927 8,492 8,508 10,426 13,368 9,836 11,522 11,042 9,826 9,503 7,952 9,303 8,128 11,580 8,784 15,384 12,601 11,863 9,396 9,404 9,590

2

Experiment

3

4,762 5,998 8,203 15,997 7,110 4,600 11,208 8,217 8,700 10,439 13,173 11,435 11,501 12,376 8,932 9,209 7,166 10,308 7,080 17,068 11,290 14,896 13,188 17,214 10,419

of the oligonucleotides were not well resolved and the figures relating to these missing. Chain length estimates based on determinations for the largest of the whole, larger than those determined for the remaining oligonucleotides. This may losses are encountered in transferring the larger oligonucleotides from the cellulose plate (18). The total radioactivity in experiments 1, 2, and 3 was, respectively, cpm.

290

SHORT

COMMUNICATIONS

by digesting the material in individual spots with pancreatic ribonuclease and quantitating the products. The pancreatic ribonuclease digestion products were fractionated and identified according to published procedures (21). A full description of the sequence-analyses will be published elsewhere. To determine the radioactivity present in each component, spots located by autoradiography were removed by the cellulose peel method of Bieleski (22), and counted in a toluene-based scintillation fluid (23). From the determination of the radioactivity present in each large oligonucleotide and in the entire digest, the total number of nucleotides in the RNA was calculated using the following formula: Total =

Number of Residues (Total (Oligonucleotide Radioactivity) Chain Length) Radioactivity in Oligonucleotide

On the assumption that the nucleotide sequence represented by each oligonucleotide is present only once in a polynucleotide chain, the polynucleotide chain lengths were calculated to be approximately 9800 nucleotides in length (Table 1). Thus, the average chain length would correspond to an RNA molecule of approximate molecular weight 3.3 x 106, a figure which is much smaller than the molecular weight estimate for the 70 S RNA. Since each of the three or four 35 S RNA subunits comprising the 70 S RNA have a molecular weight of about 3 x 106, the data, therefore, indicate that the 35 S RNAs have numerous sequence homologies. The large number of oligonucleotides examined make it unlikely that they could have all been derived from functionally related regions such as ribosome-binding sites or untranslated regions in otherwise dissimilar RNA chains. A polynucleotide chain of molecular weight 3 x lo6 barely has the coding capability to account for the known viral structural proteins and reverse transcriptase (14). If this genome also encodes other nonstructural proteins, it is likely that the

nucleotide sequences required to specify all viral proteins are contained in more than one 35 S RNA chain. In this case, the RNA subunits would possess both unique and shared nucleotide sequences. Note added in proof. At least 87% of the radioactivity in the large oligonucleotides and 97% of the total radioactivity was transferred from the cellulose acetate strips to the thin-layer plates. ACKNOWLEDGMENTS The authors thank Dr. W. K. Joklik and Marie Stone for helpful discussions and Marie Waddell and Marie Kneib for expert technical assistance. This work was supported by Grants AI-10361 and CA-12323 from the United States Public Health Service. REFERENCES

1. ROBINSON, W. S., PITKANEN, A., and RUBIN, H., Proc. Nat. Acad. Sci. USA 54, 137-144 (1965). 2. GRANBOKJLAN,

N., HUPPERT,

J., and LACOUR,

F., J.

Mol. Biol. 16, 571-575 (1966). 3. DUESBERG, P. H., Proc. Nat. Acad. Sci. USA 60, 1511-1518 4. BLAIR. C.

(1968). D., and

DUESBERG,

P.

(London) 220, 396-399 (1968). 5. ERIKSON, R. L., Virology 37, 124-131

8. 9. 10.

11. 12. 13.

14.

15.

16. 17.

Nature

(1969). A., and VIGIER, P., J. Gen. Viral. 4, 449-452 (1969). BADER, J. P., and STECK, T. L., J. Viral. 4, 459-474 (1969). FARAS, A. J., and ERIKSON, R. L., J. Viral. 4,31-35 (1969). ERIKSON, E., and ERIKSON, R. L., J. Viral. 8, 254-256 (1971). CANAANI, E., and DUESBERG, P. H., J. Viral. 10, 23-31 (1972). NICHOLS, J. L., and WADDELL, M., Nature (London) New Biol. 243, 236-238 (1973). CHEUNG, K-S., SMITH, R. E., STONE M. P., and JOKLIK, W. K., Virology 50, 851-864 (1972). CANAANI, E., HELM, K. V. D., and DUESBERG, P. H., Proc. Nat. Acad. Sci. USA 72, 401-405 (1973). VOGT, P. K., In “Possible Episomes in Eukaryotes”, (L. Silvestri, ed.), North-Holland, Amsterdam, 1973. TAYLOR, J. M., VARMUS, H. E., FARAS, A. J., LEVINSON, W. E., and BISHOP, J. M., J. Mol. Biol. 84, 217-221 (1974). SMITH, R. E., and BERNSTEIN, E. H., Appl. Microbiol. 25, 346-353 (1973). SANGER, F., BROWNLEE, G. G., and BARRELL, B. G.,

6. MONTAGNIER, 7.

H.,

L.,

GOLD%,

SHORT J. Mol.

COMMUNICATIONS

Biol. 13, 373-398 (1965). G. G., and SANGER, F., Eur. J. Bioc-hem. 11, 395-399 (1969). 19. LAI, M. M. C., and DUESBERG, P. H., Nature (London) 235, 383-386 (1972). 20. GREEN, M., and CARTAS, M., Proc. Nat. Acad. Sci. USA 69, 791-794 (1972). 18.

21.

BROWNLEE,

291

ADAMS, J. M., JEPPESEN, P. G. y., SANGER, F., and BARRELL, B. G., Nature (London) ‘223, 1009-

1014 (1969). R. L., Anal. Biochem. 12, 230-234 (1965). 23. NICHOLS, J. L., BELLAMY, A. R., and JOKLIK, W. K., Virology 49, 562-572 (1972).

22.

BIELESKI,