JOURNAL OF VIROLOGY, May 1986, p. 694-699 Vol. 58, No. 20022-538X/86/050694-06$02.00/0Copyright © 1986, American Society for Microbiology

Localization of Temperature-Sensitive Transformation Mutationsand Back Mutations in the Rous Sarcoma Virus src Gene

VALERIE J. FINCHAM AND JOHN A. WYKE*Imperial Cancer Research Fund Laboratories, St. Bartholomew's Hospital, Dominion House, London ECIA 7BE,

United Kingdom

Received 25 November 1985/Accepted 3 February 1986

Cloning and sequencing of two temperature-sensitive transforming mutations of Rous sarcoma virus revealthat their lesions are due to distinct but close single amino acid changes near the carboxy terminus of the v-srcgene product. Back mutations to wild type result from second mutations at either nearby or distant sites.

Temperature-sensitive (ts) transformation mutations ofRous sarcoma virus (RSV) have proved invaluable in corre-lating the complex phenotype of the transformed cell withthe functioning of the viral src oncogene (17). Different v-srcmutations have different consequences for the infected cell,suggesting a pleiotropism in v-src behavior (1, 3, 34), butignorance of the precise mutations has made it impossible tocorrelate these behavioral differences with defined alter-ations in the v-src gene product. Site-directed mutagenesis(5-9, 28, 29, 35) and the use of anti-peptide antibodies (13,26, 33) are now popular in analyzing the relationship be-tween v-src structure and function, but these procedures areinitially eclectic and can be coarse. A knowledge of themolecular basis of ts mutations has three benefits. (i) It

these mutants were divided, on the basis of their ability torecombine with one another to yield wild-type virus, intofour cooperative transformation groups (37, 38): memberswithin a group recombine with one another at a low butdetectable frequency while they recombine at higher fre-quency with representatives of other groups. We con-structed a coarse map of the ts mutants based on their abilityto recombine to wild type with v-src deletion mutants (11,12). This mapping, which was consistent with previousmapping based on recombination frequencies (2), has hadtwo consequences. It showed that ts mutations are dispersedwidely within src, prompting a successful search for pheno-typic differences between mutants that define different func-tional domains in v-src (30). More importantly for this paper,

LA 24LA 31

6862 7129

l lI I ynv (gp 37) ||dr| |

8706 9058

| drl lU31

PstlI I I

Psil Pstl agli Pstl BgII Pstl

FIG. 1. Salient features of the 3,096-base-pair EcoRI B fragment of the Prague strain of RSV. This fragment, from nucleotide 6144 (in env)to 9238 (in U3), encompasses the whole v-src gene and its flanking direct repeats (dr) (25). Molecular clones of this fragment from mutants,back mutants, and wild-type PrA were obtained in pAT153 after first cloning Hirt supernatant DNA from infected chick cells (10, 14), orintegrated proviral fragments from transformed chick or rat cells, in bacteriophage A vectors by standard techniques (18, 19). Major restrictionenzyme sites used in subcloning as a preliminary to sequencing are indicated. Subcloning in bacteriophage M13 (21) was followed bysequencing by the chain termination method (4, 24), (see Fig. 2 for an example). Sequences of regions of interest were confirmed by theMaxam and Gilbert method (20) after subcloning in pAT153. The bar above the line shows the predicted location of the mutations in LA24and LA31 (11). The solid arrow below the line shows the minimum region sequenced in its entirety for all the viruses studied here.

complements these other procedures, defining sites in thegene where mutations conditionally affect function. (ii) Itmay identify other regions of the gene as targets for addi-tional analyses; the propensity of ts mutants to revert to wildtype is of particular interest since the location of second-siteback mutations may indicate parts of the gene product thatinteract with one another. (iii) It provides useful informationon mutants that have been used extensively for physiologicalstudies.Our work has concentrated on a suite of ts mutants in the

v-src gene of the Prague strain of RSV, subgroup A (PrA).Soon after isolation following 5-azacytidine mutagenesis (36)

* Corresponding author.

the rough localization of the causative mutations held out thehope that they could be identified and distinguished fromother inconsequential mutations that the mutagenesis mighthave caused.We considered ts LA24 and ts LA31 ideal tests of the

feasibility of identifying ts mutations. They belong to thesame cooperative transformation group (38) and map withinabout 100 base pairs towards the 3' end of v-src (11) (Fig. 1).Both induce a markedly ts phenotype and have been used formany physiological studies. They are not leaky, so we can

isolate stable spontaneous back mutants from them, as

described previously (39). The v-src genes from LA24,LA31, three spontaneous back mutants, and the PrA progen-itor were molecularly cloned (10, 14, 18, 19) (Fig. 1). Chick

694

6269

l l

TABLE 1. Nucleotide and predicted amino acid sequence comparisons of PrA and its mutants

Change from PrC in: Presence of change in:Nucleotide

Base Amino acid' PrA LA31A 31A.1.4 31A.3.4 LA24A 24A.7b2

6517 T C L-* L (gp37,83) + + NDb ND ND ND6957 C T Noncoding (in direct repeat) + + ND ND ND ND7018 A C Noncoding + + ND ND ND ND7059 A G' Noncoding - + + + + +7082 G A Noncoding - + + +7185 A * G' H R (src,16) + + + + + +7332 C -T T T (src,69) + + + + + +7742 A-*G Y Cd(src,205) + + + + + +7749 C T R R (src,217) + - - - - -7852 G Ae A T (src,242) + + + + + +7855 A-C N H (src,243) + + + + + +7991 A Ge D G (src,288) + + + + + +8257 G A V M (src,377) - - +8561 G A G D (src,478) - + + +8567 G-A R H (src,480) - - - - + +8602 C T H Y (src,492) - - - + - +8632 G A D N (src,502) +8706 G A E E (src,526) + + + + + +

a Data in parentheses are the affected gene and amino acid.b ND, Not determined.' This change is also seen in the sequences of SRA and c-src (31, 32).d SRA and c-src differ from PrC at nucleotide 7741, substituting arginine for tyrosine (31, 32).e This change is seen in the sequences of SRA and c-src (31, 32) as well as some variants of PrC (25.)

A PrA 31A.1.4 31A. 3.4 24A.7b 2

8558 ;- AA C C T

f,I

_ -

B570

A C G T G

- / C-/ A" <

-. . T

A.. ~~~C..c

*. .._.C-cA /T

A C G T A C G T

T-3

C, Nn

C a* * T I _

.5.~ ~ 6.^

T __

TG

-""A M _s.-

oT --

AA

LA 31A

8260C T C CGTGT

8 2 54 T.

A

31A.1.4G T G C AG

AsT

Tw .

T

FIG. 2. Location of mutations and back mutations in derivatives of PrA. All sequence ladders in panel A show anti-parallel strands. Thesequences in which mutations (0) are located are indicated beside the ladders. (A) In this region (approximately nucleotides 8540 to 8630),ts LA31A (not shown) is identical in sequence to its back mutant, 31A.1.4. ts LA24A (not shown) shares with its back mutant, 24A.7b2, theG-to-A transition at nucleotide 8567 but does not display the C-to-T transition at nucleotide 8602. (B) In this region (approximately nucleotides8250 to 8265), all the viruses not shown are identical to ts LA31A.

8600 -r-GC

8604 T

B

VOL. 58, 1986 NOTES 695

give2all.org

696 NOTES

cells transfected with cloned ts mutant proviral DNA pro-duced transformants with ts morphology and protein kinaseactivity. As anticipated, transformants induced by clonedback mutant DNA lacked temperature dependence (data notshown).To determine the extent of spontaneous and mutagen-

induced variation, we sequenced (4, 20, 24) the entire EcoRIB fragment of LA31 and our current stock of wild-type PrAand compared them with the published sequence of thePrague strain of RSV, subgroup C (PrC [25]). Nucleotidenumbering of this EcoRI fragment B followed that ofSchwartz et al. (25). Table 1 summarizes the differencesbetween PrA and its derivatives and PrC.PrA and LA31 show common differences from PrC at 10

sites. Two are noncoding and another three affect the thirdbases of codons without altering coding. The other fivechanges alter the coding assignment of v-src at amino acids16, 205, 242, 243, and 288. PrA differs at two further sitesfrom PrC, LA31, LA24, and their derivatives; one of thesedifferences changes the coding of amino acid 502 of v-src.These latter changes presumably reflect variation that hasoccurred in our stock of PrA after the isolation of the tsmutants in 1971.LA31 shows an alteration at nucleotide 8561, changing

amino acid 478 of v-src from glycine to aspartic acid.Likewise, in LA24, a change at nucleotide 8567 convertsamino acid 480 from arginine to histidine (Table 1 and Fig.2). We conclude that these differences account for thetemperature sensitivity of the two mutants because they arethe only changes within v-src unique to each mutant, theyare within the region in which the mutations were located byrecombination mapping (Fig. 1), and they are very close toone another, explaining the allocation of LA31 and LA24 tothe same cooperative transformation group. Moreover, themutation in each case is a G-to-A transition, the type ofchange expected with the use of 5-azacytidine.These conclusions are strengthened by examination of

back mutations to a wild phenotype. The sequences of twoof these, LOILA31A.3.4 and LOILA24A.7b2, show that theyare identical to their parent ts viruses with one exception;both show the same C-to-T transition at nucleotide 8602,changing amino acid 492 of v-src from histidine to tyrosine(Table 1 and Fig. 2). Thus, an alteration to this amino acidappears to compensate for mutations affecting either aminoacid 478 or 480. However, this is not the only way by whichback mutations can occur, for LO/LA31A.1.4 does not differfrom parental ts LA31A in this region but shows instead amutation at nucleotide 8257, changing amino acid 377 fromvaline to methionine (Table 1 and Fig. 2).

Several interesting points emerge from this work. (i) Thesequences of LA24 and LA31 are an impressive validation ofearlier genetic studies that predicted that LA24 and LA31bear distinct but closely linked mutations (37, 38). Themutations are 6 nucleotides apart, and this remarkabledegree of linkage can therefore be discerned by examiningrecombination frequencies. Moreover, the mapping ofFincham et al. (11) of these mutations has been provenaccurate, increasing our confidence that the other 12 ts srcmutants of PrA that we have studied by genetic meansindeed have distinct mutations whose lesions span a largepart of the src gene. We suspect that the allocation of thesemutants to cooperative transformation groups does notreflect primarily the existence of hot spots for geneticrecombination between groups but gives an indication of thephysical spacing of these mutations in the src sequence.

(ii) Although LA24 and LA31 were obtained after muta-

genesis, it is noteworthy that extensive silent mutations arenot present in their genomes, and the mutations could havebeen identified without prior knowledge of their location.However, mapping studies are valuable in examining othermutants that differ from the wild type at several sites (A. W.Stoker, unpublished data). Furthermore, attempts to mapcertain ts mutants by crosses with deletion mutants haveproved equivocal (J. A. Wyke, V. J. Fincham, R. Friis, andM. Weber, unpublished data). Notably, NY68 (15) and somemutants of the GI and CU series (1, 34) recombine to thewild type with deletion mutants that lack src sequences 5' tonucleotide 8200 (approximately) but cannot do so withmutants lacking sequences 5' to nucleotide 8500 (approxi-mately). This is prima facie evidence that crucial mutationsare located between nucleotides 8200 and 8500 but thewild-type recombinants either retain some phenotypic pecu-liarities or are of irregular occurrence. The present studymakes us confident that mapping by recombination is apowerful and precise procedure, and we now consider thatthese equivocal results may be due to additional mutationselsewhere in src that also influence the phenotype of infectedcells. Recent findings (22) confirm this prediction for NY68.

(iii) The deduced amino acid sequence of our wild-typePrA differs from that of PrC (25) at several sites. Some ofthese differences are shared with the Schmidt-Ruppin strainA (SRA) of RSV and chicken c-src (31, 32) (Table 1 and Fig.3), but overall PrA resembles PrC more closely than it doesSRA. LA24 and LA31 resemble PrA in most respects, so ourwild-type clone does not represent a minor genotype in thepopulation.

(iv) The mutations in LA24 and LA31 affect amino acidsoutside the regions of pp60v-src that are most strongly con-served among tyrosine kinases, the presumed active site andATP-binding region (Fig. 3). This is not surprising, foralthough mapping has located several ts mutations in thestrongly conserved region, other mutations, as in LA29,affect pp60v-src kinase activity but map even closer to thecarboxy terminus of the protein than those of LA24 andLA31 (11, 30). This accords with the findings of Wilkersonand collaborators (35), who have studied portions of v-src byin vitro mutagenesis and propose that the carboxy terminusof the protein comprises a regulatory domain that influencesthe adjacent, conserved catalytic domain (23). The muta-tions in LA24 and LA31 are near the junction of thesepostulated domains.The altered amino acids at positions 478 and 480 are

themselves reasonably conserved in different tyrosinekinases (Fig. 3), but the significance of their alterations isunclear. Wild-type PrA also shows a change in a conservedamino acid, at position 502 (Fig. 3), but this does not affectits transforming activity. Furthermore, while it is easy toappreciate that an alteration from glycine to aspartic acid atposition 478 could result in the changes in protein stability orfunction expected in a conditional mutant, other changes inforward or back mutations are more conservative and do notconform to a discernible pattern.

(v) The significance of these mutations should be moreapparent if information on the tertiary structure of pp60v-srcis obtained. In the meantime, perhaps the most informativepart of this study is the location of spontaneous backmutations to wild type. It may only be feasible to obtain suchback mutations when the forward mutation is a single basechange, and this seems a particular advantage of ts mutantsobtained in vivo. All three back mutants retain the originalforward mutation (Fig. 2), and the compensating second-sitechanges substitute amino acids that are peculiar to these

J. VIROL.

give2all.org

NOTES 697

231V-

0 0 0 0 00o0 0 0o * 000* 0o

,SKHADGLCHR,LANVCPTSKP,QTQGLAKDAW,EIPRESLRLE,AKLGQGCFGE,VWMGTWNDTT,

291V

00 0 0000000 0 0 0 0 0 0 00000 0 000,RVAIKTLKPG,TMSPEAFLQE,AQVMKKLRHE,KLVQLYAVVS,EEPIYIVIEY,MSKGSLLDFL,

351v

00 0 0 00 0 0000 00 0 0000,KEMGKYLRL *PQLVDMAAQI,ASGMAYVOERM, NYVOH;RDLAA, NILVGENLVC, KVADFGLARLI

SR-A

PrC

PrA

31A

31A. 1.4

31A. 3.4

24A

24A. 7b2

SR-A

PrC

411

G-G-0000 000 0

,IEDNEYTARQ, GAKFPIKWTA, PEAALYGRFT,IKSDVWSFGI,ILLTELTTKGR,VPYPGMVNRE

471

R R E

00C00 000 00 00 00 00 0

,VLDOVERGYR MPCPPECPES, LHDLOMCQCWR, KDPEERPTFK, YLQAQLLPAC, VLEVAE

PrA

31A

31A. 1.4

31A. 3.4

24A

24A. 7b2

FIG. 3. Predicted sequences of the carboxy-terminal 300 amino acids of pp60v-src comparing PrC (25) with SRA (31) and PrA and its mutantderivatives. Amino acids that are conserved between v-src, v-yes, and v-fps (0) and more strongly conserved residues that are also foundin cyclic AMP-dependent protein kinase (-) are indicated (data are those of Kitamura et al. [16] and Shibuya et al. [27]). At position 295, thelysine at the ATP-binding site (A) is indicated; the putative phosphoacceptor tyrosine at position 416 (A) is also identified.

VOL. 58, 1986

SR-A

PrC

PrA

31A

31A. 1.4

31A. 3.4

24A

24A. 7b2

SR-A

PrC

PrA

31A

31A. 1.4

31A. 3.4

24A

24A. 7b2

SR-A

PrC

PrA

31A

31A. 1.4

31A. 3.4

24A

24A. 7b2

give2all.org

698 NOTES

viruses and are thus not a consequence of recombinationwith c-src. Back mutations affecting amino acid 492 mayaffect the same domain or portion of a domain as the tsforward mutations. However, the back mutation inLOILA31A.1.4. alters an amino acid on the other side of thekinase-active site. This implies a direct or indirect interac-tion between the regions around amino acids 377 and 478. Anovel interaction of this kind has been revealed after inves-tigating only three spontaneous back mutants. The facilitywith which spontaneous back mutants can be obtained froma variety of different ts src mutants suggests that they mayprovide useful and unique ways to analyze structure-function relationships in this gene.

We are grateful to David Gillespie for advice and discussion, PaulScotting for comments on the manuscript, and Andrea Sterlini forsecretarial assistance.

LITERATURE CITED1. Anderson, D. D., R. P. Beckmann, E. H. Harms, K. Nakamura,

and M. J. Weber. 1981. Biological properties of "partial"transformation mutants of Rous sarcoma virus and character-ization of their pp6Osrc kinase. J. Virol. 37:445-458.

2. Balduzzi, P., J. A. Beamand, J. R. Christensen, Y. M. Pearson,and J. A. Wyke. 1978. Provisional mapping of transformationdefective temperature-sensitive mutants of Rous sarcoma virus,p. 112-121. In S. Barlati and C. de Giuli-Morghen (ed.), AvianRNA tumour viruses. Piccin Medical Books, Padua, Italy.

3. Becker, D., R. Kurth, D. Critchley, R. R. Friis, and H. Bauer.1977. Distinguishable transformation-defective phenotypesamong temperature-sensitive mutants of Rous sarcoma virus. J.Virol. 21:1042-1055.

4. Biggin, M. D., T. J. Gibson, and G. F. Hong. 1983. Buffergradient gels and 35S label as an aid to rapid DNA sequencedetermination. Proc. Natl. Acad. Sci. USA 80:3963-3965.

5. Bryant, D., and J. T. Parsons. 1982. Site-directed mutagenesisof the src gene of Rous sarcoma virus: construction andcharacterization of a deletion mutant temperature sensitive fortransformation. J. Virol. 44:683-691.

6. Bryant, D. L., and J. T. Parsons. 1984. Amino acid alterationswithin a highly conserved region of the Rous sarcoma virus srcgene product pp6Osrc inactivate tyrosine protein kinase activity.Mol. Cell. Biol. 4:862-866.

7. Cross, F. R., E. A. Garber, and H. Hanafusa. 1985. N-terminaldeletions in Rous sarcoma virus p6Osr: effects on tyrosinekinase and biological activity, and on recombination in tissueculture with the cellular src gene. Mol. Cell. Biol. 5:2789-2795.

8. Cross, F. R., E. A. Garber, D. Pellman, and H. Hanafusa. 1984.A short sequence in the p6Os'c N terminus is required for p6Osrcmyristylation and membrane association and for cell transfor-mation. Mol. Cell. Biol. 4:1834-1842.

9. Cross, F. R., and H. Hanafusa. 1983. Local mutagenesis of Roussarcoma virus: the major sites of tyrosine and serine phosphor-ylation are dispensible for transformation. Cell 34:597-608.

10. DeLorbe, W. J., P. A. Luciw, H. M. Goodman, H. E. Varmus,and J. M. Bishop. 1980. Molecular cloning and characterizationof avian sarcoma virus circular DNA molecules. J. Virol.36:50-61.

11. Fincham, V. J., D. J. Chiswell, and J. A. Wyke. 1982. Mappingof nonconditional and conditional mutants in the src gene ofPrague strain Rous sarcoma virus. Virology 116:72-83.

12. Fincham, V. J., P. E. Neiman, and J. A. Wyke. 1980. Novelnonconditional mutants in the src gene of Rous sarcoma virus:isolation and preliminary characterization. Virology 103:99-111.

13. Gentry, L. E., L. R. Rohrschneider, J. E. Casnellie, and E. G.Krebs. 1983. Antibodies to a defined regions of pp6Osrc neutral-ize the tyrosine-specific kinase activity. J. Biol. Chem. 258:11219-11228.

14. Hirt, B. 1967. Selective extraction of polyoma DNA frominfected mouse cell cultures. J. Mol. Biol. 26:365-369.

15. Kawai, S., and H. Hanafusa. 1971. The effects of reciprocal

changes in temperature on the transformed state of cells in-fected with a Rous sarcoma virus mutant. Virology 46:470-479.

16. Kitamura, N., A. Kitamura, K. Toyoshima, Y. Hirayama, andM. Yoshida. 1982. Avian sarcoma virus Y73 genome sequenceand structural similarity of its transforming gene product to thatof Rous sarcoma virus. Nature (London) 297:205-208.

17. Linial, M., and D. Blair. 1984. Genetics of retroviruses, p.649-783. In R. Weiss, N. Teich, H. Varmus, and J. Coffin (ed.),RNA tumor viruses, 2nd ed. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

18. Loenen, W. A., and W. J. Brammar. 1980. A bacteriophagelambda vector for cloning large DNA fragments made withseveral restriction enzymes. Gene 20:249-259.

19. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

20. Maxam, A. M., and W. Gilbert. 1980. Sequencing end-labeledDNA with base-specific chemical cleavages. Methods Enzymol.65:499-560.

21. Messing, J., and J. Viera. 1982. A new pair of M13 vectors forselecting either DNA strand of double-digest restriction frag-ments. Gene 19:269-276.

22. Nishizawa, M., B. J. Mayer, T. Takeya, T. Yamamoto, K.Toyoshima, H. Hanafusa, and S. Kawai. 1985. Two independentmutations are required for temperature-sensitive cell transfor-mation by a Rous sarcoma virus temperature-sensitive mutant.J. Virol. 56:743-749.

23. Parsons, J. T., D. Bryant, V. Wilkerson, G. Gilmartin, and S. J.Parsons. 1984. Site directed mutagenesis of Rous sarcoma viruspp6Osr': identification of functional domains required for trans-formation, p. 36-42. In G. F. Vande Woude, A. J. Levine,W. C. Topp, and J. D. Watson (ed.), Cancer cells: oncogenesand viral genes. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

24. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.USA 74:5463-5467.

25. Schwartz, D. E., R. Tizard, and W. Gilbert. 1983. Nucleotidesequence of Rous sarcoma virus. Cell 32:853-869.

26. Sefton, B. M., and G. Walter. 1982. Antiserum specific for thecarboxy terminus of the transforming protein of Rous sarcomavirus. J. Virol. 44:467-474.

27. Shibuya, M., and H. Hanafusa. 1982. Nucleotide sequence ofFujinami sarcoma virus: evolutionary relationship of its trans-forming gene with transforming genes of other sarcoma viruses.Cell 30:787-795.

28. Snyder, M. A., J. M. Biship, W. W. Colby, and A. D. Levinson.1983. Phosphorylation of tyrosine-416 is not required for thetransforming properties and kinase activity of pp6o0-sr. Cell32:891-901.

29. Snyder, M. A., J. M. Bishop, J. P. McGrath, and A. D.Levinson. 1985. A mutation at the ATP-binding site of pp6oV-srcabolishes kinase activity, transformation, and tumorigenicity.Mol. Cell. Biol. 5:1772-1779.

30. Stoker, A. W., P. J. Enrietto, and J. A. Wyke. 1984. Functionaldomains of the pp60v-src protein as revealed by analysis oftemperature-sensitive Rous sarcoma virus mutants. Mol. Cell.Biol. 4:1508-1514.

31. Takeya, T., and H. Hanafusa. 1982. DNA sequence of the viraland cellular src gene of chickens. II. Comparison of the srcgenes of two strains of avian sarcoma virus and of the cellularhomolog. J. Virol. 44:12-18.

32. Takeya, T., and H. Hanafusa. 1983. Structure and sequence ofthe cellular gene homologous to the RSV src gene and themechanism for generating the transforming virus. Cell 32:881-890.

33. Tamura, T., H. Bauer, C. Birr, and R. Pipkorn. 1983. Antibod-ies against synthetic peptides as a tool for functional analysis ofthe transforming protein pp6Osrc. Cell 34:587-596.

34. Weber, M. J., and R. R. Friis. 1979. Dissociation of transfor-mation parameters using temperature-conditional mutants ofRous sarcoma virus. Cell 16:25-32.

35. Wilkerson, V. W., D. L. Bryant, and J. T. Parsons. 1985. Rous

J. VIROL.

give2all.org

VOL. 58, 1986 NOTES 699

sarcoma virus variants that encode src proteins with an alteredcarboxy terminus are defective for cellular transformation. J.Virol. 55:314-321.

36. Wyke, J. A. 1973. The selective isolation of temperature sensi-tive mutants of Rous sarcoma virus. Virology 52:587-590.

37. Wyke, J. A. 1973. Complementation of transforming functionsby temperature-sensitive mutants of avian sarcoma virus. Virol-ogy 54:28-36.

38. Wyke, J. A., J. G. Bell, and J. A. Beamand. 1975. Geneticrecombination among temperature-sensitive mutants of Roussarcoma virus. Cold Spring Harbor Symp. Quant. Biol. 39:897-905.

39. Wyke, J. A., and R. Kurth. 1978. Reversion of temperature-sensitive mutants of Rous sarcoma virus and its effect on theexpression of tumour specific surface antigen. J. Gen. Virol.40:701-704.