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Interferon-induced 56,000 Mr protein and its mRNA in human cells: molecular cloning and partial sequence of the cDNA
Abstract:
Treatment of responsive cells by interferons (IFNs) induces within a few hours a rise in the concentration of several proteins and mRNAs. In order to characterize these IFN-induced mRNA species, we have cloned in E. coli the cDNA made from a 17-18S poly(A)+ RNA of human fibroblastoid cells (SV80) treated with IFN-beta. We describe here a pBR322 recombinant plasmid (C56) which contains a 400 bp cDNA insert corresponding to a 18S mRNA species newly induced by IFN. The C56 mRNA codes for a 56,000 dalton protein easily detectable by hybridization-translation experiments. The sequence of 66 of the carboxy-terminal amino-acids of the protein can be deduced from the cDNA sequence. IFNs-alpha, beta or gamma are able to activate the expression of this gene in human fibroblasts as well as lymphoblastoid cells. The mRNA is not detectable without IFN; it reaches maximum levels (0.1% of the total poly(A)+ RNA) within 4-8 hrs and decreases after 16 hrs.
Insights
Interferons (IFNs) induce new mRNA and protein production in cells. Researchers identified a specific 18S mRNA, induced by IFNs, coding for a 56,000 dalton protein.
Area of Science:
- Molecular Biology
- Immunology
- Cell Biology
Background:
- Interferons (IFNs) are crucial signaling proteins involved in the immune response.
- IFN treatment rapidly increases cellular protein and mRNA levels.
- Characterizing these induced molecules is key to understanding IFN function.
Purpose of the Study:
- To identify and characterize novel mRNA species induced by interferon-beta (IFN-beta).
- To clone the complementary DNA (cDNA) of an IFN-induced mRNA for further analysis.
- To investigate the protein product and gene expression regulation of this IFN-responsive element.
Main Methods:
- Cloning of cDNA from IFN-beta-treated human fibroblastoid cells (SV80) into E. coli using pBR322.
- Hybridization-translation experiments to detect and analyze the protein product of the cloned cDNA.
- DNA sequencing to determine the amino acid sequence of the encoded protein.
- Northern blot analysis to assess mRNA levels under different IFN treatments and time courses.
Main Results:
- A recombinant plasmid (C56) containing a 400 bp cDNA insert for an 18S mRNA species was successfully constructed.
- The C56 mRNA codes for a 56,000 dalton protein, detectable via hybridization-translation.
- The cDNA sequence allowed deduction of 66 carboxy-terminal amino acids of the protein.
- IFNs-alpha, beta, and gamma all activate this gene's expression in both human fibroblasts and lymphoblastoid cells.
- The specific mRNA is undetectable without IFN, peaks at 4-8 hours (0.1% of poly(A)+ RNA), and declines after 16 hours.
Conclusions:
- A novel IFN-induced gene, responsive to multiple IFN types, has been identified and characterized.
- The study provides a molecular tool (C56 plasmid) for studying IFN-induced gene expression.
- The findings contribute to understanding the molecular mechanisms of interferon signaling and cellular response.