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pp60v-src tyrosine kinase is expressed and active in sarcoma-free avian embryos microinjected with Rous sarcoma virus
A R Howlett1, V C Carter, G S Martin
1Division of Cell and Molecular Biology, Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
Abstract:
Early embryonic avian tissue is resistant to transformation by Rous sarcoma virus. To determine the nature of this resistance, we examined the expression and properties of the Rous sarcoma virus transforming protein pp60v-src, in infected embryonic chicken limbs in ovo. Lysates from Rous sarcoma virus-infected limbs contained the viral structural protein p19gag, as detected by immunoblot analysis, and showed pp60v-src kinase activity in vitro. Immunoblot analysis of lysates with anti-phosphotyrosine antibodies revealed a number of phosphotyrosine-containing proteins present in lysates of Rous sarcoma virus-infected embryos but not in lysates of control, uninfected embryos. Anti-phosphotyrosine immunoreactivity was observed in frozen sections in the same cell types that expressed pp60v-src and p19gag. These studies demonstrate that pp60v-src is co-expressed with viral structural determinants in infected embryonic avian tissue. Furthermore, pp60v-src is active in ovo as a tyrosine-specific phosphotransferase, despite the apparent lack of sarcoma induction. The localization pattern of the major src gene substrate p36 (calpactin I) was compared with that of p19gag by double-label immunofluorescence and found to be generally nonoverlapping. These observations are consistent with the concept that the induction of tumors in ovo requires complementation between viral determinants and host factors. These host factors, which may be critical substrates of pp60v-src, are subject to developmental regulation in the avian embryo.
Insights
Early embryonic avian tissues resist Rous sarcoma virus transformation. The transforming protein pp60v-src is active in ovo, but tumor induction requires host factors, suggesting developmental regulation.
Area of Science:
- Oncology
- Virology
- Developmental Biology
Background:
- Early embryonic avian tissues exhibit resistance to Rous sarcoma virus (RSV) transformation.
- Understanding the mechanisms behind this resistance is crucial for cancer research.
Purpose of the Study:
- To investigate the expression and activity of the RSV transforming protein pp60v-src in infected embryonic chicken limbs in ovo.
- To elucidate the factors contributing to the resistance of embryonic avian tissue to RSV-induced transformation.
Main Methods:
- In ovo infection of embryonic chicken limbs with Rous sarcoma virus.
- Immunoblot analysis to detect viral protein p19gag and pp60v-src kinase activity.
- Immunoblot analysis with anti-phosphotyrosine antibodies to identify phosphoproteins.
- Double-label immunofluorescence to compare the localization of pp60v-src substrates and viral proteins.
Main Results:
- RSV-infected embryonic limbs expressed both p19gag and active pp60v-src kinase.
- Phosphotyrosine-containing proteins were detected in infected embryos but not in controls.
- pp60v-src and p19gag were co-expressed in infected embryonic tissues.
- The major src substrate, p36 (calpactin I), showed non-overlapping localization with p19gag.
Conclusions:
- pp60v-src is active as a tyrosine-specific phosphotransferase in ovo, despite the lack of sarcoma induction.
- Tumor induction in ovo likely requires complementation between viral factors and developmentally regulated host factors.
- Host factors, potentially critical substrates of pp60v-src, are subject to developmental regulation in avian embryos.