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[Mutations in Escherichia coli pmp and htpR genes stabilize the products of foreign gene expression]
Abstract:
The half lives of mRNA for Escherichia coli chloramphenicol-acetyltransferase, Bacillus amyloliquefaciens alpha-amylase and human leucocyte interferon were measured in E. coli cells by molecular RNA.DNA hybridization. The effect of mutation in pnp gene, coding polynucleotide phosphorylase, on the stability of these mRNA was studied. The half life of interferon mRNA increases from 25 to 90 s in the pnp mutant, resulting in an increase of interferon accumulation. The stability of interferon in E. coli cells depends on the htpR gene, controlling the heat shock response. The yields of leucocyte interferons alpha-2, alpha I-1 and fibroblast interferon beta increase ten times in htpR mutants. Thus, by using pnp and htpR mutants it is possible to enhance considerably the eukaryotic gene expression in bacterial cells.
Insights
Mutations in the pnp and htpR genes significantly enhance the stability and accumulation of eukaryotic mRNA, like human interferon, in bacterial cells. This finding offers a promising strategy for boosting eukaryotic gene expression in bacteria.
Area of Science:
- Molecular biology
- Microbial genetics
- Biotechnology
Background:
- Understanding mRNA stability is crucial for controlling gene expression.
- Bacterial systems are often used for producing eukaryotic proteins.
- The stability of foreign mRNA in bacteria can be a limiting factor for expression yields.
Purpose of the Study:
- To investigate the impact of specific gene mutations on mRNA half-life in Escherichia coli.
- To determine if these mutations can enhance the expression of eukaryotic genes in bacteria.
- To explore the role of polynucleotide phosphorylase (pnp) and heat shock response (htpR) genes in mRNA stability.
Main Methods:
- Measuring mRNA half-lives using molecular RNA.DNA hybridization in E. coli.
- Analyzing the effect of mutations in the pnp gene on mRNA stability.
- Assessing the influence of the htpR gene mutation on interferon mRNA stability and accumulation.
- Quantifying the yields of different types of interferons in mutant strains.
Main Results:
- The half-life of human leucocyte interferon mRNA increased from 25 to 90 seconds in pnp mutants.
- Interferon accumulation increased in pnp mutants.
- The htpR gene mutation, linked to heat shock response, further enhanced interferon stability.
- Yields of leucocyte interferons (alpha-2, alpha I-1) and fibroblast interferon beta increased tenfold in htpR mutants.
Conclusions:
- Mutations in pnp and htpR genes can significantly stabilize eukaryotic mRNA in E. coli.
- These genetic modifications lead to a substantial increase in the production of eukaryotic proteins, such as interferons.
- Targeting bacterial genes involved in RNA degradation and stress response presents a viable method for enhancing eukaryotic gene expression in microbial hosts.