The structure and protein kinase activity of proteins encoded by nonconditional mutants and back mutants in the sec gene of avian sarcoma virus

We have characterizedsrc proteins encoded by approximately 30 nonconditional transformation-defective mutants of avian sarcoma virus (ASV) and by several back mutants which reestablish a transformed phenotype. We used gel electrophoresis of immunoprecipitated proteins labeled with³²PO₄ or [³⁵S]methionine to assess size, stability, and phosphorylation; partial digestion with staphylococcal V8 protease to determine structure; and an immune complex assay to measure protein kinase activity. The mutants were all isolated as phenotypic revertants of the B31 line of B77-ASV transformed rat cells, each revertant cell bearing a single provirus without appreciable deletions, as described in the accompanying report (Varmuset al., 1980). In several instancesm the mutant proteins were examined both in the revertant rat cells and in chicken cells infected with transformation-defective viruses rescued from the nonpermissive rat cells. In addition, secondary mutations to restore a transformed phenotype (back mutations) occurred in some cases, in the original rat cells and/or chicken cells infected with rescued viruses. Three categories of mutants were identified by this survey. The largest group (Class I) encodedsrc proteins of normal size (60,000M_r); these proteins were hypophosphorylated and exhibited little or no protein kinase activity.Class II mutants displayed immunoprecipitablesrc proteins of less than normal size. In three cases, the shortsrc related proteins were mapped to the amino terminus of wild-type pp60^src and may be the result of nonsense mutations; in two cases, the short proteins were mapped to the car?yl terminus. Most of Class II mutants lacked protein kinase activity, but the 45,000M_r protein in line 000 exhibited moderate levels of activity, thereby mapping the enzymatically active site to the car?yl terminal three-fourths of pp60^src. The smallest group of mutants (Class III) did not produce detectablesrc proteins. Some of the mutant proteins behaved differently in permissive and nonpermissive hosts; in particular, the product of mutant L produced fusiform transformation and was highly phosphorylated and associated with wild-type levels of protein kinase activity in chicken cells, but was nontransforming, hypo-phosphorylated, and associated with low levels of protein kinase activity in rat cells. In all cases, back mutation to a transformed phenotype was accompanied by a restoration of wild-type (or near wild type) levels of protein kinase activity, further documenting the functional significance of the enzymatic activity. Some of the back mutants, however, encoded proteins of atypical size, either smaller or larger than pp60^src. The active proteins larger than pp60^src ranged up to 68,000M_r in size and were altered at or near the amino terminus. In one case (a retransformed derivative of the Class II revertant 000), the generation of a functionalsrc protein of 68,000M_r coincided with the appearance of an insert of ca. 200 base pairs into the ASV provirus, within or adjacent to the coding region for the amino terminus ofsrc. The diversity of reagents, both mutants and back mutants, derived from the single provirus in B31 cells indicates that this system will be useful for correlation of functional and structural attributes ofsrc.     

Characterization of the avian sarcoma virus protein p60src.

The intracellular distribution of the avian sarcoma virus (ASV) protein, p60^src, has been investigated in transformed chicken and hamster cells by radioimmunoprecipitation of fractionated cells and by immunofluorescent staining of fixed cells with monospecific antiserum to p60^src. The src protein was found exclusively in the cytoplasmic fraction in both a soluble and an insoluble form. Furthermore, we show that the antigenicity of p60^src was extremely heat-labile in vitro relative to Pr76, the precursor to the internal structural proteins of ASV. Antibody directed against p60^src which is present in serum from rabbits bearing tumors induced by the Schmidt-Ruppin (subgroup D) strain of ASV does not recognize p60^src from cells transformed by other ASV strains including Prague C, B77, and Bryan (RAV-50). Re-evaluation of the precipitation of p60^src from NY68-transformed cells has led to the conclusion that there is no reduction in the synthesis or antigenicity of p60^src when cells are cultured under conditions nonpermissive for transformation by this mutant virus.     

Endogenous proviruses of random-bred chickens and ring-necked pheasants: analysis with restriction endonucleases

We have examined, by digestion with restriction endonucleases and nucleic acid hybridization, sequences homologous to avian sarcoma virus (ASV) DNA in DNA from 18 random-bred chickens of the brown leghorn and brown nick flocks and 8 ring-necked pheasants. Both species have sequences related to the replicative genes (gag, pol, andenv) and to the transforming gene (src) of ASV. The disposition of these sequences in random-bred chickens is reminiscent of the situation in inbred white leghorn flocks; the sequences related togag, pol, andenv appear to reside in structures which closely resemble proviruses of the endogenous chicken virus RAV-O, and thesrc-related sequences appear to be a cellular gene (or genes). The number of endogenous proviruses present in the random-bred flocks is highly variable, and there are proviruses present at positions in the genomes of the random-bred birds different from those described for white leghorns. The endogenous ASV-related sequences in ring-necked pheasants fall into the same two categories; sequences related to the replicative genes of ASV probably reside in proviruses, and thesrc-related sequences in a cellular gene (or genes). However, the endogenous pheasant viruses are clearly distinct from those of chickens both by analysis with restriction endonucleases and by hybridization. These observations support the hypothesis that cellularsrc (c-src) has had a separate evolutionary history from the endogenous proviruses, which apparently arise by germ line infections. The endogenous viruses of chickens and pheasants, while clearly related, appear to have undergone significant independent evolution, which suggests that the frequency with which these viruses achieve a successful germ line infection across species boundaries is low compared with the rate of successful germ line infections within a species.     

Structural and nonstructural proteins of strain Colburn cytomegalovirus

The growth of most Rous sarcoma viruses (RSV) is severely restricted on MSB-1 cells (a line of chicken T lymphoblasts) in comparison to growth on chicken embryo fibroblast (CEF). Nonconditional transformation defective mutants of RSV from which the complete src region has been deleted (td RSV) are not subject to growth restriction. We examined the formation and integration of RSV and td RSV in MSB-1 cells following high multiplicity infection. Nearly equivalent quantities of the linear form of unintegrated RSV and td RSV DNA were formed in these cells during the first 10 hr after infection. Linear RSV DNA from MSB-1 cells could not be distinguished from linear RSV from CEF by restriction endonuclease analysis and by previously described transfection assays (P. E. Neiman, C. McMillin-Helsel, and G. M. Cooper, 1978, Virology 89,360–371). Beyond 10 hr after infection, and with progressive cell growth in the MSB-1 cultures, the level of RSV linear DNA rapidly decreased. Presumptive circular RSV DNA was detected only transiently, and at very low levels, about 15 hr after infection. Association of RSV DNA with high-molecular-weight chromosomal DNA, i.e., integration, was not detected in this study. In contrast, nearly constant levels of td RSV unintegrated linear DNA and, after 20 hr, circular DNA persisted in MSB-1 cells for at least 7 days after infection. Integration of td RSV proviral DNA was inefficient, occurring in only about 5% of MSB-1 cells (even at very high multiplicities of infection) in the first round of infection, and in 25–40% of cells by 3 days after infection. Almost all MSB-1 cells containing td RSV DNA produced virus. Analysis of eight nonconditional transformation defective mutants of RSV which retain the src region to different extents showed that all of these mutants replicated to the same normal titer on MSB-1 cells as on CEF without further deletion of the src region. Two temperature sensitive src mutants that thermal inactivation of the scr gene on MSB-1 cells at both 35° and 41°, indicating that thermal inactivation of the src gene product could not abrogate the replication block. These studies clearly demonstrate that the presence of the src region in RSV impedes the formation and/or integration of provirus in some types of host cells.     

Continuous production of Rous sarcoma virus free of transformation defective virus in clones of established RSV-transformed quail cells

Quail embryo fibroblasts were infected with a Schmidt-Ruppin strain RSV × chf recombinant virus. Virus-transformed cells were established as a permanent line and then cloned in methyl cellulose. Out of 140 clones isolated four clones were capable of indefinite growth. These clones were examined for (i) production of sarcoma and td virus particles, (ii) number of integrated virus genome equivalents, and (iii) deletions of the src gene in the provirus. We found that the clones yield about 106 focus-forming units of the sarcoma virus per milliliter of the culture medium. No td virus could be detected by plating of the virus at the endpoint dilution and no 35 S td virus RNA but only 38 S sarcoma virus RNA was found in virions. Hybridization kinetic studies indicated that three different clones contain about 2 virus genome equivalents, and one clone contains about 4 virus genome equivalents per diploid cell. Upon transfection the proviruses of different clones generated sarcoma viruses and no td viruses. Finally digestion with EcoRI restriction endonuclease released in all four clones a 1.9 × 106-dalton fragment characteristic of the complete src gene, while no 0.8 × 10⁶-dalton fragment characteristic of a td provirus could be detected. We concluded that the clones of RSV-transformed quail cells contain only nondefective sarcoma proviruses and produce from these proviruses nondefective focus-forming virions in the absence of any segregant td virions.     

Specific restriction of avian sarcoma viruses by a line of transformed lymphoid cells.

MSB-1 cells are a line of transformed chicken lymphoid cells derived from tumors induced by Marek's disease viruses and free of exogenous avian leukosis viruses (ALV). They can be infected by ALV of subgroups A and C including transformation-defective (td) deletion mutants of avian sarcoma viruses (ASV). In terms of virus titers in supernatant culture medium, proportion of virus-producing cells, and levels of viral RNA detected by hybridization with a cDNA probe, infection by td ASV of MSB-1 cells was indistinguishable from infection of chicken embryo fibroblasts. In contrast, wild type ASV was restricted in its growth on MSB-1 cells. Different clones of ASV varied in their restriction by all these parameters of viral growth by factors of 10^?1 to 10^?4 Studies of a severely restricted viral clone showed equal quantities of hybridizable viral DNA in Hirt supernatant fractions of both fibroblasts and MSB-1 cells at 10 hr after high multiplicity infection, and transfection assays indicated infectious viral DNA in both cell types. Viral DNA largely disappeared from Hirt supernatant fractions of MSB-1 cells by 48 hr after infection, and sarcoma virus-specific DNA was not detected in Hirt pellet fractions from MSB-1 cells at levels found in comparably infected fibroblasts. Infectious ASV DNA, while easily detected in fibroblasts, could not be detected on MSB-1 cells at 48 hr or later times after infection. Because replication of td ASV does not appear restricted in MSB-1 cells, the failure of ASV DNA to integrate normally in these cells seems to be related to the presence of src sequences in the viral genome.     

Novel localization of pp60src in Rous sarcoma virus-transformed rat and goat cells and in chicken cells transformed by viruses rescued from these mammalian cells

We have investigated by indirect immunofluorescence and subcellular fractionation the intracellular location of pp60^src in RSV-transformed mammalian cells and in CEF cells transformed by virus rescued from these cells. Two independently derived cell lines were examined: RR1022 cells isolated from an in vivo sarcoma induced in an Amsterdam rat by infection with SR-RSV-D; and Pcl, cells isolated from a soft agar colony of normal goat skin fibroblasts transformed in vitro by SR-RSV-D. Transforming viruses (RSV-RR and RSV-Pcl) were rescued from RR1022 and Pcl cells by fusion with CEF cells. Immunofluorescence microscopy showed association of pp60^src with the nuclear envelope and the juxtanuclear reticular membranes in the transformed mammalian cells and in CEF cells transformed by the rescued viruses, in contrast to the plasma membrane localization of pp60^src seen in SR-RSV-transformed CEF cells. Results of subcellular fractionation by differential centrifugation and fractionation of particulate fractions by equilibrium centrifugation in discontinuous sucrose gradients were in agreement with the differences in pp60^src distribution observed by immunofluorescence microscopy. Although the mammalian cell lines were independently derived, pp60^srcs isolated from RR1022 and Pcl cells both lacked amino-terminal 21- and 18-kilodalton [³⁵S]methionine S. aureus V8 protease peptides found in SR-RSV-D pp60^src. Proteolytic peptides identical to those of pp60^src from the mammalian cells were obtained from pp60^src proteins isolated from rescued virus-transformed CEF cells, suggesting that the alteration in the amino-terminal half of the src protein represents a stable change, and that an alteration in the primary structure of pp60^src is responsible for the altered intracellular membrane localization of pp60^src in these cells.     

Reversion and induction of Rous sarcoma virus expression in virus-transformed baby hamster kidney cells

All revertant subclones of Schmidt-Ruppin virus transformed BHK cells retained the viral genome. Virus could be rescued by Sendai virus-mediated cell fusion with susceptible chicken cells; however, the frequency of virus rescue from the revertant subclones was much lower than the frequency of virus rescue from transformed subclones. Treatment of revertant subclones with 5BUdR before cell fusion and virus rescue led to a 10-fold increase in the observed rescue frequency.     

Transformation by adenovirus early region 2A temperature-sensitive mutants and their revertants

H5ts107 is a temperature-sensitive mutant of adenovirus type 5 whose alteration maps in the structural gene for the viral DNA binding protein. Temperature-independent revertants of this mutant that form plaques at 39° in HeLa cells have been isolated. These revertants fall into two classes: (1) ts⁺ revertants for growth and plaque formation at 39° in both HeLa cells and 293 cells, a human cell line transformed by type 5 adenovirus; (2) ts⁺ for growth and plaque formation at 39° in HeLa cells, but temperature-sensitive (ts) for growth and plaque formation in 293 cells. The frequency of rat cell transformation by H5tsl07 was fivefold higher than that of wild-type adenovirus. A class 1 revertant, r(tsl07)127, transformed cells at the wild-type virus frequency while a class 2 revertant, r(tsl07)202, retained the ability to transform cells at the higher frequency. When the XhoI-C DNA fragment from these viruses, which contains the left end 15.5% of the viral genome, was employed to transform rat cells, then Ad5 wt, H5tsl07, r(tsl07)127, and r(ts107)202 DNAs all transformed with approximately equal frequencies.     

Second site mutations in the N-terminus of the major capsid protein (VP5) overcome a block at the maturation cleavage site of the capsid scaffold proteins of herpes simplex virus type 1.

VP5, the major capsid protein of herpes simplex virus type 1 (HSV-1), interacts with the C-terminal residues of the scaffold molecules encoded by the overlapping UL26 and UL26.5 open reading frames. Scaffold molecules are cleaved by a UL26 encoded protease (VP24) as part of the normal capsid assembly process. In this study, residues of VP5 have been identified that alter its interaction with the C-terminal residues of the scaffold proteins. A previously isolated virus (KUL26-610/611) was used that encoded a lethal mutation in the UL26 and UL26.5 open reading frames and required a transformed cell line that expresses these proteins for virus growth. The scaffold maturation cleavage site between amino acids 610 and 611 was blocked by changing Ala-Ser to Glu-Phe, which generated a new EcoRI restriction site. Revertant viruses, that formed small plaques on nontransformed cells, were detected at a frequency of 1:3800. Nine revertants were isolated, and all of them retained the EcoRI site and therefore were due to mutations at a second site. The second site mutations were extragenic. Using marker-transfer techniques, the mutation in one of the revertants was mapped to the 5' region of the gene encoding VP5. DNA sequence analysis was performed for the N-terminal 571 codons encoding VP5 for all of the revertant viruses. Six of the nine revertants showed a single base pair change that caused an amino acid substitution between residues 30 and 78 of VP5. Three of these were identical and changed Ala to Val at residue 78. The data provide a partial map of residues of VP5 that alter its interaction with scaffold proteins blocked at their normal cleavage site. The yeast two-hybrid system was used as a measure of the interaction between mutant VP5 and scaffold molecules and varied from 11% to nearly 100%, relative to wild-type VP5. One revertant gave no detectable interaction by this assay. The amount of UL26 encoded protease (VP24) in B capsids for KUL26-610/611 and for revertants was 7% and 25%, respectively, relative to the amount in capsids for wild-type virus. The lack of retention of the viral protease in the mutant virus and a fourfold increase for the revertants suggest an additional essential function for VP24 in capsid maturation, and a role in DNA packaging is indicated.     

Temperature-sensitive kinase activity associated with various mutants of avian sarcoma viruses which are temperature sensitive for transformation

Antisera against the transforming gene product of avian sarcoma virus (ASV), called pp60^src, were raised in tumor-bearing rabbits by inoculation of a high dose of the avian sarcoma virus Schmidt-Ruppin strain of subgroup D (SR-D) into newborn animals. Four mutants of ASV with temperature-sensitive defects for transformation have been investigated for temperature sensitivity of the sarcoma gene-associated protein kinase activity in comparison to that of their wild-type parents. The inactivation rate of the protein kinase from the mutants MI 100, OS122, OS538, and NY68 was two-to threefold faster than that of the respective parents which belong to various subgroups of the Schmidt-Ruppin strain, SR-B, SRD, and SR-A. During heat inactivation, dephosphorylation of ³²P-labeled pp60^src immune precipitated from mutant virus-infected cells was not parallel to the inactivation of the kinase. The amount of [³⁵S]methionine-labeled pp60^src precipitated from mutant- and wild type-infected cells was not sensitive to heat treatment. The kinase activity associated with pp60^src of mutant- and wild type-infected cells was protected from inactivation by the presence of sera from tumor-bearing rabbits (TBR) during the heating procedure. Protein kinase activity of MI100 increased up to fourfold during this process, while with other mutant and wild-type kinase activities such a curing by TBR-sera was also observed but not to the same extent. Immune complexes obtained from transformed cells treated with TBR-serum and Staphylococcus aureus were used as a source of kinase(s) to phosphorylate exogenously added cell lysates as targets. Alternatively, [γ-³²P]ATP was added to cell lysates to allow phosphorylation of polypeptides present in the lysates. This procedure resulted in phosphorylation of proteins with molecular weights of 60K, 35K, and 23K which were absent from the normal or leukosis-virus-infected controls. Several proteins of high molecular weight, one of them of 150K, were enhanced. Treatment of these phosphoproteins with TBR-serum resulted in precipitation of the 60K and 23K proteins but not of the 150K and 35K phosphoproteins.     

Loss of proviral DNA sequences in a revertant of Kirsten sarcoma virus-transformed murine fibroblasts.

A previously described revertant cell line (K-BALB SR1212), derived as a single cell clone from a clonal line of murine fibroblasts (K-BALB) transformed by a nonproductive infection with the Kirsten strain of murine sarcoma virus, has normal morphology and growth kinetics and, unlike the transformed parent cell line, lacks a sarcoma virus that can be rescued. We report here that this reversion correlates with low to undetectable levels of expression of cellular Ki-MSV-specific RNA and a reduction of proviral sequences in the cell DNA to a level equivalent to that found in the uninfected BALB cells with a normal phenotype. The data indicate that phenotypic reversion has occurred as a consequence of the loss of part or all of the sarcoma provirus, either by chromosomal rearrangement or provirus excision.     

Reversion to normal phenotype induced by SV40 in a spontaneously transformed malignant Chinese hamster cell line

By using a selection procedure that excluded the transforming effect of SV40, reversions to several properties of normal phenotype were for the first time obtained in a transformed Chinese hamster cell line after SV40 infection. The value of induction to recovery of contact inhibition was typical for SV40-induced reverse gene mutations. Thirteen of 15 isolated revertant clones were T-antigen positive, thus synthesizing the product of viral oncogene. Therefore, in the majority of clones reversion occurred in spite of the presence of viral transforming protein. Dot hybridization revealed the presence of SV40 DNA in all revertants including those expressing no T antigen. The virus rescued from one T-antigen positive and two negative clones proved to be infectious. Reversion to contact inhibition was followed by reversion as regards serum requirements and growth in soft agar. However, in all cases reversion was partial. Karyologic analysis of revertant clones showed that six clones maintained the hypodiploid karyotype of the parental clone, six revertants were near-tetraploid, and one was near triploid. The possible events underlying the SV40-induced reversions to normal phenotype and the role of virus-induced mutations in viral carcinogenesis are discussed.     

Different mechanisms for morphologic reversion of a clonal population of murine sarcoma virus-transformed nonproducer cells.

Morphologic revertants of a clonal line of BALB/3T3 nonproductively transformed by Kirsten murine sarcoma virus were isolated by selection in methylcellulose semisolid suspension medium. Each revertant clone was obtained from a separate mutagenized cell culture in an attempt to isolate the widest spectrum of genetically altered cells. The newly isolated revertants were indistinguishable from BALB/3T3 in morphology and in vitro growth properties. One group, like a previously reported series (Greenberger, J. S., Anderson, G. R., and Aaronson, S. A. (1974). Cell2, 279–286.) was shown to contain reversibly altered sarcoma viral genomes. These cell lines expressed high levels of sarcoma viral RNA, and wild-type transforming virus became rescuable at a time when cells in the population spontaneously retransformed. This occurred at low frequencies ranging from one in 10⁶ to 10⁸ cell generations. There was no evidence of complementation or recombination between the altered sarcoma viruses present in these revertant lines. A second group of revertants was very stable to retransformation and failed to demonstrate rescuable sarcoma virus. These cell lines contained no more sarcoma viral RNA or DNA than uninfected BALB/3T3 indicating that these revertants arose from loss of the sarcoma virus genome. Thus, the present studies indicate that both alteration and loss of sarcoma viral information are reproducible causes of reversion to the nontransformed state in this system.     

Organization of the endogenous proviruses of chickens: implications for origin and expression

Eleven of the endogenous proviruses of white leghorn chickens have been mapped with restriction endonucleases and specific nucleic acid hybridization reagents. The restriction maps of these endogenous proviruses have been compared with restriction maps of avian sarcoma virus (ASV) and Rous-associated virus O (RAV-O), an endogenous virus which is spontaneously released by cells from certain lines of chickens. Endogenous proviruses have the same basic structure as proviruses acquired by exogenous infection; the gene order is the same in the provirus as in viral RNA, and the ends of the provirus form a characteristic direct repeat which contains sequences derived from both ends of viral RNA. The endogenous proviruses can thus be described “cell DNA-3′5′-gag-pol-env-3′5′-cell DNA,” where 3′ and 5′ denote sequences homologous to the 3′ and 5′ ends of viral RNA. The endogenous proviruses of chickens are more closely related to RAV-O than to ASV, based on restriction maps and on hybridization with reagents specific for the 3′ ends of RAV-O and ASV. However, all but two of the endogenous proviruses lack at least one of the twoSstI sites in RAV-O DNA (see the preceding paper) and can thus be distinguished from RAV-O by digestion withSstI. One of the two exceptions,ev-2, is found in the DNA of line 7₂ and line 100 chickens and is genetically linked to the production of RAV-O. The only other provirus (which we call B) having both theSstI sites in RAV-O DNA was seen only once in a line 100 sample. Of the nine remaining elements, six had large deletions. Three proviruses (ev-4,ev-6, and an element we call A) are missing the left 3′5′ repeat and have sustained deletions extending varying distances intogag orpol. One of these,ev-6, is associated with the gs^?chf⁺ phenotype; the phenotype can be explained from the structure of the provirus.ev-3 has both terminal repeats intact but has sustained a deletion near thegag-pol boundary. This provirus is associated with the gs⁺chf⁺ phenotype and the structure of the provirus could account for the peculiar RNA and protein associated with this phenotype. We have also found two elements which apparently consist of sequences present only in the 3′5′ terminal repeat unit, with no other associated virus specific sequences. Such structures might arise by homologous recombination between the terminal repeats of a normal provirus.     

Mapping of nonconditional and conditional mutants in the src gene of Prague strain Rous sarcoma virus

Restriction endonuclease EcoRI digestion of the viral DNA of 12 nonconditional transformation defective (td) mutants of Prague strain Rous sarcoma virus (PR-RSV) has divided these mutants into two groups. Five mutants possess an EcoRI B (src gene-containing) fragment of the same size as that from wild type PR-RSV and thus these mutants have no detectable diminution in the transforming src gene. The other 7 mutants bear deletions of 1.0 to 1.8 kilobases in the 3.2-kilobase EcoRI B fragment. The extents of these deletions have been mapped using a number of restriction endonucleases and by comparing these results with studies on the nucleotide sequence of src(Czernilovsky et al., Nature (London)287, 198–203, 1980) we conclude that the td mutants have deleted sequences at the 5′ end of src, and in some cases also in regions between src and env, leaving intact at least some 3′ src sequences. These td mutants recombine in differing patterns with 14 temperature-sensitive (ts) src gene mutants. This enables many of the ts mutations to be localized in limited regions of src, 10 of them being clustered in the 3′ 40% of the gene, the remaining four bearing at least one mutation in the 5′ 60% of src. A nonconditional src gene mutant that transforms cells to a fusiform as opposed to round cell morphology (td SF/LO 104) also possesses a lesion that maps in the 5′ 60% of the src gene.     

Cell-free translation of purified avian sarcoma virus src mRNA

Avian sarcoma virus 21 S RNA, purified by hybridization from virus-infected cells, was translated in a cell-free system. The major product of translation was a protein of 60,000 daltons. This protein was the same as authentic pp60^src, the product of the ASV src gene, when compared by electrophoretic mobility in polyacrylamide gels, immunological reactivity and partial protease digestion. These findings confirm that the 21 S ASV RNA serves as mRNA for pp60^src. Furthermore, pp60^src is the only major product of translation of the src gene and is apparently synthesized without a cleavable signal sequence.