Identification of tryptic peptides unique to a 110,000-molecular weight polyprotein encoded by the T-8 isolate of murine leukemia virus.

Mink cells nonproductively-infected with the weakly-transforming T-8 isolate of murine leukemia virus (MuLV) express a 110,000 mol wt polyprotein designated T-8 P110. By immunoprecipitation analysis, T-8 P110 is shown to contain AKR-MuLV amino terminal gag gene-specific components (p15, p12) but to lack p30, p10, gp70, and p15(E) antigenic determinants. These observations are further substantiated by tryptic peptide analysis indicating T-8 P110 to share approximately six lysine-containing tryptic peptides with AKR-MuLV Pr65gag, and none with AKr-MuLV Pr82env. Furthermore, of seven methionine-containing T-8 P110 tryptic peptides, at least four can be conclusively shown not to be present in either AKr-MuLV Pr180gag/pol or Pr82env. A clonal mink cell line nonproductively infected by T-8, and expressing high levels of P110, although not morphologically transformed, is shown to lack elevated levels of tyrosine-specific protein kinase activity and reduction of epidermal growth factor binding sites characteristic of cells transformed by many other RNA-transforming viruses. These findings argue either that the T-8 viral genome contains acquired cellular sequences encoding a portion of P110, or that T-8 P110 represents an inphase deletion of AKR-MuLV Pr180gag/pol with extensive posttranlational modification and that an as yet unidentified protein is responsible for T-8 associated transformation.

Anemia- and polycythemia-inducing isolates of Friend spleen focus-forming virus. Biological and molecular evidence for two distinct viral genomes.

Two distinct clones of Friend spleen focus-forming virus (SFFV), differing in their erythroleukemic potential, are described. These isolates have been cloned free of their associated helper viruses and shown to be replication-defective. Both SFFV isolates have been rescued from rat fibroblast nonproducer cell clones with cloned replication-competent viruses, F-MuLVA and F-MuLVP, obtained from the anemia- or polycythemia-inducing isolates of Friend virus complex, respectively. These rescued viruses induce a rapid proliferative disease associated with the appearance of macroscopic spleen foci and splenomegaly. In addition, each is subject to regulation by the W, Steel (Sl), and Fv-2 host gene loci. These two isolates of SFFV can, however, be distinguished by both biological and molecular criteria. Friend SFFVP induces a rapid polycythemia associated with the appearance of large numbers of erythropoietin (EPO)-independent erythroid colony-forming cells in the marrow and spleen. In contrast, SFFVA induces a rapid anemia associated with a progressive decrease in the number of EPO-dependent erythroid colony-forming cells in marrow, and a rapid increase in the number of EPO-dependent erythroid colony-forming cells in spleen. Furthermore, the nature of the disease induced by the two isolates of SFFV is independent of the Friend helper virus: SFFVP, rescued from a nonproducer cell clone with either F-MuLVA or F0MuLVP, induced a polycythemic transformation, whereas SFFVA, rescued with either F-MuLVA or F-MuLVP, induced an anemic transformation. The two Friend SFFV isolates can also be discriminated on the basis of translational products encoded by their gag and env genes: SFFVP encodes the amino-terminal gag-gene protein p15, whereas SFFVA encodes the gag-gene proteins p15, p12, and p30. In addition, the SFFV isolates encode nonidentical 55,000-mol wt env gene-related proteins that can be distinguished by analysis of their methionine-containing tryptic peptides.

Transformation induced by Abelson murine leukemia virus involves production of a polypeptide growth factor.

Rat embryo fibroblasts transformed by Abelson murine leukemia virus (MuLV) produce and release a transforming growth factor (TGF). Production of this factor is correlated with a tyrosine-specific protein kinase that is functionally active and is associated with the major Abelson MuLV gene product, P120. Transformation-defective mutants of Abelson MuLV do not transform cells, do not have their virus coded transforming gene product phosphorylated in tyrosine, and do not induce TGF production. Abelson MuLV-induced TGF morphologically transforms cells in culture, competes with 125I-labeled epidermal growth factor (EGF) for binding to cell receptors, and induces phosphorylation of tyrosine acceptor sites in the 160,000-dalton EGF membrane receptor. After purification to homogeneity, Abelson virus-induced TGF migrates as a single polypeptide with an apparent size of 7400 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Evidence that the phl gene encodes a 160,000-dalton phosphoprotein with associated kinase activity.

In chronic myelocytic leukemia, the human c-abl oncogene is translocated from chromosome 9 to a region on chromosome 22 designated as the breakpoint cluster region (bcr) (A. de Klein, A. Guerts van Kessel, G. Grosveld, C. R. Bartram, A. Hagemeyer, D. Bootsma, N. K. Spurr, N. Heisterkamp, J. Groffen, and J. R. Stephenson, Nature (London) 300:765-767, 1982; J. Groffen, J. R. Stephenson, N. Heisterkamp, A. de Klein, C. R. Bartram, and G. Grosveld, Cell 36:93-99.) Abnormal c-abl homologous mRNA and protein have been detected in the leukemic cells of patients with chronic myelocytic leukemia (E. Canaani, D. Stein-Saltz, E. Aghai, R. P. Gale, A. Berrebi, and E. Januszewicz, Lancet 1:593-595, 1984; S. J. Collins and M. T. Groudine, Proc. Natl. Acad. Sci. USA 80:4813-4817, 1983; R. P. Gale and E. Canaani, Proc. Natl. Acad. Sci. USA 81:5648-5652, 1984; J. B. Konopka, S. M. Watanabe, J. W. Singer, S. J. Collins, and O. N. Witte, Proc. Natl. Acad. Sci. USA 82:1810-1814, 1985). The abnormal mRNA represents a chimeric transcript consisting of 5' bcr and 3' c-abl sequences (G. Grosveld, J. Verwoerd, T. van Agthoven, A. de Klein, K. L. Ramachandran, N. Heisterkamp, K. Stam, and J. Groffen, Mol. Cell. Biol. 6:607-616, 1986; E. Shtivelman, B. Lifshitz, R. B. Gale, and E. Canaani, Nature (London) 315:550-554, 1985; K. Stam, N. Heisterkamp, G. Grosveld, A. de Klein, R. S. Verma, M. Coleman, H. Dosik, and J. Groffen, N. Engl. J. Med. 313:1429-1433, 1985). In the present study, we demonstrated that the abnormal c-abl protein is a fusion protein. In addition, the normal gene encompassing bcr sequences was shown to encode a 160,000-dalton phosphoprotein with an associated serine or threonine kinase activity. We propose that this gene be designated phl, reserving the term bcr for the region within the phl gene encompassing the Ph' translocation breakpoints.

Preparation and characterization of monoclonal antibodies specific for human transforming growth factor alpha.

A series of monoclonal antibodies (mAbs) against transforming growth factor alpha (TGF alpha) have been produced. The generation of these reagents, as well as their biochemical and immunochemical characterization is described. TGF alpha peptides, mutant recombinant TGF alpha proteins and two-site immunoradiometric assays were used to identify the epitopes recognized by each antibody. This approach has allowed the specific localization of immunodominant domains on the molecule. Certain mAbs were found to be useful for selected procedures. mAb 134A-2B3 was used for immunoblotting both the precursor and mature forms of TGF alpha from conditioned media of tumor cells. One mAb 189-2130.1, which reacted with the carboxyl terminal seventeen amino acids, was able to block TGF alpha binding to the EGF receptor. mAb 213-4.4 was used for immunohistochemical detection of TGF alpha in fixed tumor cells. mAbs 137-178 and 134A-2B3 were used to develop a two-site immunoradiometric immunoassay which was sensitive to 1 ng ml-1 and detected TGF alpha from a variety of tumor cells. A series of mAbs such as these could prove useful in studying the biochemical properties as well as the immunochemical localization of TGF alpha in normal tissues and tumors.

Immunological identification and distribution of parathyroid hormone-like protein polypeptides in normal and malignant tissues.

Monoclonal and polyclonal antibodies recognizing human parathyroid hormone-like protein (PTHLP) have been produced using a series of recombinant and synthetic PTHLP peptides. These antibodies have been used to develop a two-site immunometric enzyme immunoassay which detects PTHLP[1-87] and PTHLP[1-141] but not PTH. The immunoassay detected PTHLP in extracts of squamous carcinomas and normal tissues at concentrations from 7-515 ng PTHLP[1-87]/mg protein. Immunoblotting of the extract which showed the highest immunoreactivity, a squamous carcinoma of the lung from a patient with hypercalcemia, revealed a major band having an apparent mol wt of 26,500 and several other higher mol wt bands. Similar polypeptides were observed by immunoblotting cell extracts from a cell line, SCaBER, which secretes immunoreactive PTHLP into its medium and also from tumors in nude mice derived from this cell line. Chaotropic agents did not alter the immunoblotting pattern, and antibodies to three different epitopes of PTHLP recognized these bands, indicating PTHLP expression in the extracts. Immunohistochemical staining of normal human tissue with these antibodies revealed several PTHLP-containing tissues and confirmed the results of the immunoassay, suggesting a paracrine role for PTHLP. Staining was observed in several neoplastic tissues including squamous cell carcinomas, lung carcinoma, bladder carcinoma, osteogenic sarcoma, and adenocarcinoma of the colon.

Synthesis of a gene encoding parathyroid hormone-like protein-(1-141): purification and biological characterization of the expressed protein.

PTH-like proteins (PTHLP), which are associated with humoral hypercalcemia of malignancy, have recently been purified. Isolation of their corresponding cDNAs has revealed that they are derived from a single gene. In this report a synthetic gene encoding PTHLP-(1-141), a 141-amino acid protein corresponding to the most abundant PTHLP cDNA detected in human tumors, was expressed in bacteria and purified to homogeneity. Recombinant (r) PTHLP-(1-141) migrates with an aberrantly high mol wt on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, presumably as a result of its unusually basic pI. rPTHLP-(1-141), like PTH, induced hypercalcemia in rats, caused release of 45Ca from fetal rat bones, and stimulated the synthesis of cAMP by rat osteosarcoma cells and canine renal membrane preparations. A comparison of the abilities of rPTHLP-(1-141) and bovine PTH-(1-34) to stimulate cAMP synthesis indicated rPTHLP-(1-141) to be 5-fold more potent in the osteosarcoma assay, while nearly 30-fold less active in the renal membrane adenylate cyclase assay. Although 100-fold less potent than bovine PTH-(1-34) in promoting bone resorption, rPTHLP-(1-141) was a potent calcemic factor in vivo, inducing a rise in serum calcium from 10.4 to 14.5 mg/dl when infused into rats at 1.3 micrograms/h. These results support previous assumptions that PTHLP is the humoral factor responsible for humoral hypercalcemia of malignancy. In addition, they suggest substantial differences between PTHLP and PTH in the regulation of calcium homeostasis.

Effect of rearing environment and dietary zinc oxide on the response of group-housed weaned pigs to enterotoxigenic Escherichia coli O149 challenge.

A 2 × 2 factorial experiment was conducted to determine the effects of rearing environment (indoor (In) v. outdoor (Out)) and dietary zinc oxide (ZnO) supplementation (0 (-Zn) v. 3100 (+Zn) mg/kg feed) on the response of weaned pigs to a challenge infection with enterotoxigenic Escherichia coli (ETEC). Pigs from the two rearing environments were weaned onto trial diets at 4 weeks of age, moved into conventional accommodation and infected 3 days later with 109 CFU ETEC per os. Faecal ETEC shedding was determined before and after challenge. After 7 days of ETEC infection, all pigs were euthanized for gut lactic acid bacteria (LAB)-to-coliform ratio, pH and small intestine morphological measurements. Both ZnO and outdoor rearing reduced ETEC excretion, and these effects were additive. Outdoor rearing increased small intestine and colon tissue weight. ZnO increased villus height and goblet cell number in the upper small intestine, LAB-to-coliform ratio (through reduced coliforms) in the lower small intestine and proximal colon, and improved growth performance. There were interactive effects of rearing environment and ZnO supplementation on upper small intestine villus height and daily gain, as outdoor rearing conferred advantages on these variables only with ZnO dietary supplementation. Daily gains were 233, 174, 277 and 347 (s.e.m. 27.2) g/day for the In - Zn, Out - Zn, In + Zn and Out + Zn, respectively. These results suggest different, but complementary mechanisms of intestinal health and performance in outdoor-reared pigs and those offered ZnO supplemented diets. The results indicate that the benefits of ZnO to the weaned pig extend beyond suppression of ETEC and appear mediated through altered development of the small intestine mucosa.

Does the newly weaned piglet select a zinc oxide supplemented feed, when given the choice?

An experiment was conducted to examine whether weaned piglets would display preference for a food containing a pharmacological level of zinc oxide (ZnO). A total of 60 piglets were weaned at 7.8 kg ± 0.14 (s.e.m.) and 27.8 ± 0.11 days of age into eight mixed sex groups of seven or eight piglets per pen. Groups were balanced for litter origin, weaning weight and sex. Piglet feeding behaviour was constantly recorded by a multi-spaced feeding behaviour recording system (Leeds University Feeding Behaviour System) in each pen. Each pen of pigs was offered ad libitum access to two different foods (16.2 MJ digestible energy, 16 g lysine/kg), which differed only in the level of ZnO supplementation: unsupplemented (U) or supplemented (Z; ZnO 3100 mg/kg). Both foods contained a basal level of zinc (100 mg/kg). Feeding time was recorded for each individual at each trough. Piglets were weighed at weaning and at 7 and 13 days thereafter. The experiment ran for 13 days. Any piglet observed with post-weaning scour (Y) was recorded and treated appropriately whereas healthy piglets were categorised as N (no scour). Preference for a food was defined as being significantly different from 50% of total feed intake or time spent feeding. There was no difference between piglet numbers selecting each food as their first meal. However, within the first 24 h, piglets preferred (P < 0.001) food U, spending only 36.3% (32.2 to 40.5; 95% confidence interval) of feeding time at food Z. Throughout the experiment, piglets showed aversion (P < 0.001) to food Z, consumption being 8.9% (5.1 to 13.6) and 15.7% (8.9 to 23.9) of total intake in weeks 1 and 2, respectively. Individual piglets showed their preference for food U with only 16.6% (14.6 to 18.5) and 21.8% (19.6 to 24.0) of feeding time spent on food Z in weeks 1 and 2, respectively. Scouring piglets did not show any difference in feeding behaviour from healthy piglets in either week. Average piglet gain (of all piglets) was low, at 0.039 ± 0.03 and 0.272 ± 0.04 kg/day in weeks 1 and 2, respectively. Given a choice, weaned piglets showed a clear preference for the food U even when exhibiting post-weaning scour. It can be concluded that the newly weaned, naïve, piglet is not able to recognise a food with clear health and performance benefits but selected the food U due to the reduced palatability of the food Z.

Abelson murine leukemia virus P120: identification and characterization of tyrosine phosphorylation sites.

Tryptic peptides containing two major in vivo P120gag-abl tyrosine phosphorylation acceptor sites were identified, phosphorylated in vitro, and purified to homogeneity. The tyrosine site in peptide a is localized at a position six residues distal to its trypsin cleavage site, whereas the tyrosine acceptor site in peptide b is at residue seven. A third peptide, c, contains an amino-terminal phosphotyrosine residue: phosphorylation of this latter peptide only occurs to a significant extent in vivo.

Interferon activity produced by translation of human interferon messenger RNA in cell-free ribosomal systems and in Xenopus oöcytes.

Translation of messenger RNA isolated from poly(rI)-poly(rC)-induced human fibroblasts in cell-free ribosomal systems and in Xenopus oocytes resulted in the production of biologically active proteins that had the properties of human fibroblast interferon. The translation in the oocytes was much more efficient, giving approximately 500 times higher titers of interferon activity than the cell-free systems. A control messenger RNA isolated from noninduced human fibroblasts, did not code for interferon synthesis in these systems. Both messenger RNA preparations stimulated [3H]amino-acid incorporation into trichloroacetic acid-insoluble material. The radioactive products and their immunoprecipitates were electrophoresed on polyacrylamide gels under denaturing conditions. The products resulting from the translation of the control (uninduced) messenger RNA in oocytes contained a major protein of approximately 45,000 molecular weight. The messenger RNA isolated from poly(rI)-poly(rC)-induced cells stimulated the synthesis of an additional 25,000 molecular weight protein that electrophoresed in the same position as human fibroblast interferon. These results suggest that human fibroblast interferon was synthesized by the translation of its messenger RNA in Xenopus oocytes and in cell-free ribosomal systems.

New mammalian transforming retrovirus: demonstration of a polyprotein gene product.

A new acute transforming type C retrovirus was isolated from mice inoculated with a virus stock obtained by iododeoxyuridine induction of methylcholanthrene-transformed C3H/10T1/2 mouse cells. This virus, designated 3611-MSV, transforms embryo fibroblasts and epithelial cells in culture and induces fibrosarcomas in vivo. 3611-MSV is replication defective, requiring a type C helper virus for propagation both in vitro and in vivo. By using endpoint transmission of 3611-MSV to MMCE C17 mouse and FRE 3A rat cells, several nonproductively transformed clonal cell lines have been derived. Pseudotype virus stocks obtained from such clones transform cells in vitro, are highly oncogenic in vivo, and exhibit host range and serological properties that are characteristic of their helper virus component. Analysis of viral antigen expression in 3611-MSV-transformed cells has led to the demonstration of a 90,000-molecular-weight (Mr) polyprotein and a 75,000-Mr probable cleavage product, both containing the amino-terminal murine leukemia virus gag gene proteins p15 and p12. In contrast to gene products of many previously described mammalian transforming viruses, 3611-MSV-encoded polyproteins lack detectable protein kinase activity, and 3611-MSV-transformed cells resemble chemically transformed cell line C3H/MCA-5, from which 3611-MuLV was originally derived, in that they do not exhibit elevated levels of phosphotyrosine. By using molecular hybridization the 3611-MSV transforming gene was found to be distinct from previously described mammalian cellular oncogenic sequences, including c-ras, c-abl, c-fes, c-fms, c-sis, and c-mos.

The role of cell membrane in the antiviral effect of interferon.

The mechanism of interferon action in human fibroblasts has been studied by use of both antisera to human fibroblast interferon and the antisera to the surface of human fibroblast cell. The anti-interferon serum completely neutralized the antiviral effect of human fibroblast interferon. Interferon antiserum prevented the intracellular antiviral state from developing when added to the medium of the cells in which interferon synthesis had already been induced by poly (I-C). This suggests that development of the antiviral state involves interferon interaction with the external part of the producing cell. Treatment with the serum directed against the surface of human fibroblast cells failed to inhibit the antiviral activity of human interferon in these cells. In addition, the effect of gangliosides on the antiviral activity of human interferon was studied and it was found that human interferon binds to gangliosides and that this interaction leads to inactivation of the antiviral effect of interferon. Pretreatment of human fibroblasts with gangliosides had no effect on the sensitivity of these cells to exogenous interferon.

Cells nonproductively transformed by Abelson murine leukemia virus express a high molecular weight polyprotein containing structural and nonstructural components.

Cell clones nonproductively transformed by the replication-defective Abelson strain of murine leukemia virus (AbLV) were analyzed for type C viral antigen expression by competition immunoassay. AbLV-transformed mink non-producer lines were found to express a 110,000- to 130,000-molecular weight polyprotein containing murine leukemia virus gag proteins p15 and p12 covalently linked to nonstructural AbLV-coded component(s) of around 80,000-100,000 molecular weight. This polyprotein lacked detectable antigenic cross-reactivity with other virion-coded gag gene proteins such as p30, p10, the viral reverse transcriptase (RNA-dependent DNA polymerase), or the major viral envelope glycoprotein, gp70. By analogy to earlier data on feline and avian sarcoma viruses, these results suggest that a portion of this polyprotein might represent the AbLV src gene product and that in translation it is initially linked in precursor form to gag structural proteins. Superinfection of mink cells nonproductively transformed by AbLV--with either a wild mouse amphotropic type C virus isolate, 4070-A, or with the endogenous cat virus, RD114--led to production of pseudotype virus containing high concentrations of the AbLV-coded precursor polyprotein.

Feline sarcoma virus P115-associated protein kinase phosphorylates tyrosine. Identification of a cellular substrate conserved during evolution.

The Gardner strain of feline sarcoma virus (FeSV) has previously been shown to encode as its major translational product, a phosphoprotein of molecular weight (Mr) = 115,000 (P115) with an associated protein kinase activity and probable transforming function. In the present study, we demonstrate that this enzyme specifically phosphorylates tyrosine and that Gardner FeSV-transformed mink and rat cells exhibit constitutively higher levels of phosphotyrosine than nontransformed control cells. In addition, we identify a Mr = 150,000 cellular substrate for the FeSV P115-associated protein kinase activity. This latter protein, designated P150, is expressed in cells of a number of mammalian species including cat, mink, dog, rat, mouse and human, possesses broadly reactive interspecies antigenic determinants, and exhibits binding affinity for FeSV P115. Although P150 exhibits an associated protein kinase activity, this activity differs from the FeSV P115-associated enzyme in that it phosphorylates serine and threonine rather than tyrosine. On the basis of immunologic criteria and tryptic peptide analysis, the possibility that P150 is structurally related to Gardner FeSV P115 is excluded. As a consequence of its affinity for FeSV P115, P150 is efficiently incorporated into FeSV pseudotype virions, providing a source for its purification and preparation of antisera for study of its expression in mammalian cells.

Human transforming growth factors induce tyrosine phosphorylation of EGF receptors.

Cultured cell lines of human tumour origin as well as cells transformed by various RNA tumour viruses secrete low molecular weight polypeptide transforming growth factors (TGFs). In addition to competing with epidermal growth factor (EGF) for binding to its cellular receptor, TGFs can transform morphologically fibroblast and epithelial cells in culture. In view of accumulating evidence that tyrosine phosphorylation activity is associated with the transforming genes of various tumour viruses, we determined whether phosphotyrosine levels were elevated in these human tumour cells. We show here that TGFs produced by human tumour cells induce phosphorylation of specific tyrosine acceptor sites in the 160,000-molecular weight (160 K) EGF receptor.

Differences in mechanisms of transformation by independent feline sarcoma virus isolates.

The Gardner and Snyder-Theilen isolates of feline sarcoma virus (FeSV) have previously been shown to encode high-molecular-weight polyproteins with a transforming function and an associated tyrosine-specific protein kinase activity. Cells transformed by these viruses exhibited morphological alterations, elevated levels of phosphotyrosine, and a reduced capacity for binding epidermal growth factor. In addition, polyproteins encoded by both of these FeSV isolates bound to, and phosphorylated tyrosine acceptor sites within, a 150,000-molecular-weight cellular substrate (P150). McDonough FeSV-transformed cells resembled Gardner and Snyder-Theilen FeSV transformants with respect to morphological changes and a reduced capacity for epidermal growth factor binding. in contrast to the other two FeSV isolates, however, McDonough FeSV encoded as its major translational product a high-molecular-weight polyprotein with probable transforming function but without protein kinase activity detectable under similar assay conditions. Moreover, total cellular levels of phosphotyrosine remained unaltered in McDonough FeSV-transformed cells, and the major McDonough FeSV polyprotein translational product lacked binding affinity for P150. These findings argue for differences in the mechanisms of transformation by these independently derived FeSV isolates.

Involvement of a high-molecular-weight polyprotein translational product of Snyder-Theilen Feline sarcoma virus in malignant transformation.

The previously described high-molecular-weight polyprotein major translational product of the Snyder-Theilen strain of feline sarcoma virus (FeSV) was shown to possess protein kinase activity with specificity for tyrosine acceptor sites. Cells transformed by Snyder-Theilen FeSV exhibited constitutively elevated levels of phosphotyrosine and a concomitant reduction in epidermal growth factor (EGF) binding sites. By endpoint cloning in microtiter plates, a number of transformation-defective (tf) mutants of the Snyder-Theilen strain of FeSV were isolated. Mink cells nonproductively infected by such mutants were morphologically nontransformed, failed to grow in soft agar, bound EGF as efficiently as control mink cells, and lacked rescuable transforming virus. Although the level of expression of the major viral polyprotein translational product in td mutant-infected clones was comparable to that of wild-type (wt) transformants, the polyprotein in mutant clones lacked detectable protein kinase activity and total cellular phosphotyrosine levels were not elevated significantly above control values. Of a large number of wt Snyder-Theilen FeSV-transformed mink cell clones isolated, the majority were found to revert to a nontransformed morphology upon continuous passage in cell culture. Such nontransformed variants, as well as a Gardner FeSV-transformed mink cell revertant, lacked detectable polyprotein expression and exhibited levels of phosphotyrosine and EGF binding similar to those of control mink cells. These findings provide strong evidence favoring the involvement of the Snyder-Theilen FeSV-encoded high-molecular-weight polyprotein and its associated tyrosine-specific protein kinase activity in transformation.