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A cellular enhancer of retrovirus gene expression in embryonal carcinoma cells
Abstract:
Murine embryonal carcinoma (EC) cells are refractory to infection by retroviruses because retroviral long terminal repeat (LTR) enhancers have little activity in EC cells. A previous report described the isolation of clonal EC cell lines that express the integrated neomycin-resistance gene (neo) linked to the Moloney murine leukemia virus LTR. The expression of the neo gene was explained by a cis-acting mechanism [Taketo, M., Gilboa, E. & Sherman, M. I. (1985) Proc. Natl. Acad. Sci. USA 82, 2422-2426]. From one such EC cell line, we isolated the flanking cellular sequence 5' to the proviral genome, ligated it to various test constructs, and transfected into the parental EC cells. The cellular sequence increased expression of the LTR-linked neo gene significantly, in a manner independent of its orientation and position. The neo mRNA was initiated at the bona fide promoter of the LTR. By deletion analyses, we defined a region of DNA essential for the enhancer activity and determined its sequence. This region contains distinctly characteristic stretches as well as some similarity to various viral and cellular enhancers. Thus the LTR-linked neo gene is expressed because the provirus is integrated in the vicinity of this enhancer that is active in undifferentiated EC cells.
Insights
Murine embryonal carcinoma cells resist retroviruses due to low enhancer activity. A newly discovered cellular enhancer near proviral integration activates gene expression in these cells.
Area of Science:
- Developmental Biology
- Molecular Virology
- Gene Regulation
Background:
- Murine embryonal carcinoma (EC) cells exhibit resistance to retroviral infection.
- This resistance is attributed to the limited activity of retroviral long terminal repeat (LTR) enhancers in EC cells.
- Previous studies identified EC cell lines expressing a neomycin-resistance gene (neo) linked to a Moloney murine leukemia virus LTR, suggesting a cis-acting mechanism.
Purpose of the Study:
- To investigate the mechanism behind the expression of the LTR-linked neo gene in EC cells.
- To identify and characterize cellular sequences that can enhance retroviral LTR activity in EC cells.
Main Methods:
- Isolation of cellular DNA sequences flanking the proviral genome in an EC cell line expressing the neo gene.
- Ligation of these sequences to test constructs and transfection into parental EC cells.
- Deletion analyses to define the essential region for enhancer activity and DNA sequencing.
Main Results:
- A specific cellular sequence, located 5' to the proviral genome, significantly increased the expression of the LTR-linked neo gene.
- This enhancer activity was independent of orientation and position and initiated neo mRNA at the LTR promoter.
- Deletion analyses identified a DNA region crucial for enhancer function, containing unique and similar motifs to other viral and cellular enhancers.
Conclusions:
- Undifferentiated EC cells possess an enhancer element active in their vicinity.
- The expression of the LTR-linked neo gene in previously isolated EC cell lines is due to the integration of the provirus near this cellular enhancer.
- This finding explains the previously observed cis-acting mechanism for neo gene expression in EC cells.