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Interferons as gene activators. Cloning of the 5' terminus and the control segment of an interferon activated gene
Abstract:
Treatment of cells with interferons induces various mRNAs and the corresponding proteins. We have described previously the isolation of a mouse cDNA clone (cDNA clone 202) which specifies an mRNA whose level is increased 20-fold in beta-interferon-treated Ehrlich ascites tumor cells. The increase is a consequence of an increased rate of transcription. The mRNA encodes a 56,000-dalton protein. We report here the isolation of a genomic clone including the 5' terminus of the 202 gene with the interferon-responsive region. Experiments involving primer extension and protection from cleavage by S1 nuclease revealed the existence of multiple 5' termini of 202 mRNAs in Ehrlich ascites tumor and Ltk- cells. Treatment with beta-interferon increased the level of these 202 mRNAs with different 5' termini nonuniformly. A 0.8-kilobase DNA segment from the 202 gene (including its 5' flanking region and its 5'-terminal exon) was ligated to the chloramphenicol acetyltransferase gene, and the resulting construct was transfected into mouse Ltk- cells. Treatment of these cells with beta-interferon increased the expression of the chloramphenicol acetyltransferase gene 5-10-fold. Within the first, untranslated exon of the 202 gene, we found a 29-nucleotide long sequence that is partially homologous to sequences which occur upstream from interferon-inducible human HLA and metallothionein IIA genes (Friedman, R. L., and Stark, G. R. (1985) Nature 314, 637-639).
Insights
Interferon treatment upregulates specific gene expression through transcriptional control. Researchers identified an interferon-responsive region in the 202 gene, demonstrating its role in gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- Immunology
Background:
- Interferons (IFNs) are crucial signaling molecules that modulate cellular responses, including the induction of specific mRNAs and proteins.
- Previous work identified mouse cDNA clone 202, encoding a 56-kDa protein, with a 20-fold increase in mRNA levels upon beta-interferon treatment due to enhanced transcription.
Purpose of the Study:
- To isolate and characterize the genomic clone of the 202 gene, focusing on its 5' terminus and interferon-responsive elements.
- To investigate the transcriptional regulation of 202 gene expression by beta-interferon.
Main Methods:
- Isolation of a genomic clone containing the 5' region of the 202 gene.
- Analysis of 202 mRNA 5' termini using primer extension and S1 nuclease protection assays.
- Reporter gene assays involving transfection of a construct containing the 202 gene's 5' regulatory region linked to the chloramphenicol acetyltransferase (CAT) gene.
Main Results:
- Multiple 5' termini for 202 mRNAs were identified in different cell types.
- Beta-interferon treatment led to a non-uniform increase in the levels of 202 mRNAs with varying 5' ends.
- A DNA segment from the 202 gene's 5' region conferred interferon inducibility to the CAT reporter gene, showing a 5-10 fold increase in expression.
- A conserved 29-nucleotide sequence in the 5'-untranslated exon showed homology to regulatory elements in other interferon-inducible genes.
Conclusions:
- The 5' region of the 202 gene contains regulatory elements responsible for interferon-mediated transcriptional induction.
- This regulatory region, including a conserved sequence, plays a key role in the cellular response to interferons.