Related Experiment Videos
Expression of the mouse mammary tumor virus ORF gene in cultured cells
1Swiss Institute for Experimental Cancer Research, Epalinges.
Abstract:
We have recently shown that expression vectors harboring the open reading frame of the long terminal repeat region of mouse mammary tumor virus direct the synthesis of a product which acts as a superantigen in transgenic mice. The detection of the ORF protein has been hampered by the extremely low levels of expression observed in these mice, as estimated from the low levels of specific mRNA. To study the properties of the ORF protein, we attempted its expression in different cell types in culture. The experiments performed in yeast show that the ORF gene product is a glycoprotein of approximately 45 kDA. As expected from the derived primary sequence, the unglycosylated product made in the presence of tunicamycin has a molecular weight of 36 kDA. No secretion of the glycosylated protein was observed. Curiously, the full-length molecule was made in lower amounts than a truncated version which contains only the C-terminal half of the protein. Transfection experiments in different mammalian cells suggest that high expression of the ORF protein might have an adverse effect on survival of cells in culture.
Insights
Researchers expressed the mouse mammary tumor virus open reading frame (ORF) protein in yeast, identifying it as a 45 kDa glycoprotein. High expression in mammalian cells may negatively impact cell survival.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Mouse mammary tumor virus (MMTV) open reading frame (ORF) protein acts as a superantigen in transgenic mice.
- Detection of the MMTV ORF protein is challenging due to very low expression levels in vivo.
Purpose of the Study:
- To investigate the properties of the MMTV ORF protein through expression in various cell types.
- To characterize the MMTV ORF gene product and its expression patterns.
Main Methods:
- Expression of the MMTV ORF gene in yeast and mammalian cells.
- Analysis of protein size and glycosylation using tunicamycin.
- Assessment of protein secretion and expression levels.
Main Results:
- The MMTV ORF gene product expressed in yeast is a glycoprotein of approximately 45 kDa.
- The unglycosylated form, produced in the presence of tunicamycin, has a molecular weight of 36 kDa.
- No secretion of the glycosylated protein was observed, and higher expression levels in mammalian cells may be toxic.
Conclusions:
- The MMTV ORF protein is a glycoprotein that is not efficiently secreted.
- High-level expression of the MMTV ORF protein can adversely affect mammalian cell survival.