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Mouse cells contain two distinct ras gene mRNA species that can be translated into a p21 onc protein
Abstract:
The Kirsten (Ki) and Harvey (Ha) strains of murine sarcoma virus encode a 21,000-dalton protein (p21 ras) which is the product of the transforming gene of these viruses. Normal cells express low levels of p21 ras encoded by cellular genes (Ki-ras and Ha-ras) homologous to the Ki and Ha murine sarcoma virus transformation genes. A bone marrow-derived mouse cell line, 416B, has been shown to express unusually high levels of p21 ras. In this manuscript, we investigated the molecular biology of p21 ras gene expression in 416B and other normal mouse cells. We identified four distinct polyadenylated and polysome-associated RNAs, two related to Ki-ras and two to Ha-ras. The levels in 416B cells of the two Ki-ras RNAs, sized 5.2 and 2.0 kilobases, were both elevated approximately 25-fold over levels found in normal mouse cells; there was no corresponding change in 416B cells in the levels of the two Ha-ras RNAs. We partially purified the two Ki-ras mRNAs and separated them by velocity sedimentation in sucrose density gradients. Both the 5.2- and 2.0-kilobase mRNAs could be translated in vitro into p21 ras. These results show that a cellular onc protein can be translated from two distinct cellular mRNA species.
Insights
High levels of the oncogenic protein p21 ras in 416B cells are due to a 25-fold increase in two specific Ki-ras messenger RNAs. These distinct Ki-ras mRNAs can both be translated into the p21 ras protein.
Area of Science:
- Molecular Biology
- Oncogenes
- Gene Expression
Background:
- Murine sarcoma viruses encode a 21,000-dalton protein (p21 ras) from their transforming genes.
- Normal cells express low levels of p21 ras via homologous cellular Ki-ras and Ha-ras genes.
- The 416B mouse cell line exhibits unusually high p21 ras expression.
Purpose of the Study:
- Investigate the molecular basis of p21 ras gene expression in 416B cells.
- Characterize the specific ras gene expression alterations in this cell line.
- Determine if distinct mRNA species can produce the p21 ras protein.
Main Methods:
- RNA isolation and analysis from 416B and normal mouse cells.
- Identification and quantification of polyadenylated, polysome-associated RNAs.
- In vitro translation assays using partially purified Ki-ras mRNAs.
Main Results:
- Four distinct polyadenylated, polysome-associated RNAs (two Ki-ras, two Ha-ras) were identified.
- 416B cells showed a ~25-fold increase in two Ki-ras RNAs (5.2 and 2.0 kb).
- No significant change in Ha-ras RNA levels was observed in 416B cells.
- Both 5.2 kb and 2.0 kb Ki-ras mRNAs were translated in vitro to produce p21 ras.
Conclusions:
- Elevated p21 ras in 416B cells is linked to increased expression of specific Ki-ras mRNAs.
- Two distinct cellular Ki-ras mRNA species can independently translate into the p21 ras oncoprotein.
- This highlights the potential for differential mRNA usage in oncogene protein production.