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Two distinct sequence elements mediate retroviral gene expression in embryonal carcinoma cells
Abstract:
Moloney murine leukemia virus (M-MuLV) and M-MuLV-derived retroviral vectors are not expressed in early mouse embryos or in embryonal carcinoma cells. M-MuLV-derived mutants or M-MuLV-related variants which transduce the neomycin phosphotransferase gene can, however, induce drug resistance in embryonal carcinoma cells with high efficiency. In this study we investigated the sequences critical for retroviral gene expression in two different embryonal carcinoma cell lines, F9 and PCC4. We show that two synergistically acting sequence elements mediate expression in embryonal carcinoma cells. One of these is located within the U3 region of the viral long terminal repeat, and the second one is in the 5' untranslated region of the retrovirus. The latter element, characterized by a single point mutation, affects the level of stable RNA in infected cells, suggesting a regulatory mechanism similar to that of human immunodeficiency virus in human T cells.
Insights
Moloney murine leukemia virus (M-MuLV) gene expression is blocked in early embryos but can be restored in embryonal carcinoma cells. Two specific DNA sequence elements are critical for enabling M-MuLV gene expression in these cells.
Area of Science:
- Retroviral gene expression
- Developmental biology
- Molecular genetics
Background:
- Moloney murine leukemia virus (M-MuLV) and its derived vectors show limited expression in early mouse embryos and embryonal carcinoma (EC) cells.
- M-MuLV-derived mutants can confer drug resistance to EC cells, indicating potential for gene transfer.
Purpose of the Study:
- To identify the specific DNA sequences responsible for M-MuLV gene expression in F9 and PCC4 embryonal carcinoma cell lines.
- To elucidate the regulatory mechanisms governing retroviral gene expression in early developmental contexts.
Main Methods:
- Analysis of M-MuLV gene expression in F9 and PCC4 EC cells.
- Identification and characterization of critical sequence elements within the M-MuLV genome.
- Mutation analysis to assess the impact of specific sequences on viral RNA levels.
Main Results:
- Two distinct sequence elements synergistically enhance M-MuLV expression in EC cells.
- One element is located in the U3 region of the viral long terminal repeat (LTR).
- A second element in the 5' untranslated region, affected by a single point mutation, influences stable RNA levels.
Conclusions:
- Specific sequence elements in the M-MuLV LTR and 5' UTR are essential for gene expression in embryonal carcinoma cells.
- The identified regulatory mechanism may share similarities with human immunodeficiency virus (HIV) regulation in T cells.
- These findings provide insights into the control of retroviral gene expression during early development.