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Expression of a reverse transcriptase activity in a cell line established from peritoneal macrophages of mice treated
I Demirhan1, H Massalha, A Chandra
1Laboratorium für Molekularbiologie (ZBC), Johann Wolfgang Goethe, Universität Frankfurt am Main, Germany.
Abstract:
We have established a cell line from peritoneal macrophages of mice treated intraperitoneally with N-methyl-N-nitrosourea. The present communication describes the identification of an RNA-dependent DNA-polymerase activity in a particulate fraction from supernatants of the cell culture. This activity is similar to retroviral Reverse Transcriptase (RT) based on its template specificity and ionic preference. The proof of the retrovirus-like nature of RT was obtained by ultracentrifugation of the pelleted proteins secreted in the medium on a sucrose gradient. The main RT activity was obtained in fractions of 1.14-1.16 g/ml densities, which are comparable to those of type C retroviruses. The presented data support constitutive expression of the retrovirus gene in a chemically transformed cell line.
Insights
Researchers identified retroviral Reverse Transcriptase (RT) activity in a chemically transformed mouse cell line. This suggests constitutive expression of retroviral genes in cancer cells, offering insights into viral gene expression.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Chemically induced cell lines are valuable models for studying gene expression.
- Retroviral Reverse Transcriptase (RT) is crucial for retroviral replication.
- Understanding RT activity in transformed cells can reveal insights into oncogenesis.
Purpose of the Study:
- To identify and characterize RNA-dependent DNA-polymerase activity in a mouse peritoneal macrophage cell line induced by N-methyl-N-nitrosourea.
- To determine if this activity resembles retroviral Reverse Transcriptase (RT).
- To investigate the potential constitutive expression of retroviral genes in chemically transformed cells.
Main Methods:
- Established a cell line from peritoneal macrophages of mice treated with N-methyl-N-nitrosourea.
- Isolated a particulate fraction from cell culture supernatants.
- Assayed for RNA-dependent DNA-polymerase activity.
- Performed ultracentrifugation on sucrose gradients to analyze secreted proteins.
Main Results:
- Identified significant RNA-dependent DNA-polymerase activity in the cell culture supernatant.
- Characterized the activity as similar to retroviral Reverse Transcriptase (RT) based on template specificity and ionic preference.
- Demonstrated that the main RT activity banded at densities (1.14-1.16 g/ml) comparable to type C retroviruses via sucrose gradient ultracentrifugation.
Conclusions:
- The chemically transformed mouse cell line constitutively expresses a retrovirus-like gene.
- The identified RNA-dependent DNA-polymerase activity is consistent with Reverse Transcriptase (RT).
- These findings support the hypothesis of endogenous retroviral gene expression in N-methyl-N-nitrosourea-induced transformed cells.