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Updated: Jul 16, 2026

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

Reverse transcriptases: intron-encoded proteins found in thermophilic bacteria.

Bernie Ng¹, Sunil Nayak, Moreland D Gibbs

¹Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109, Australia.

|March 17, 2007

Summary

Researchers identified reverse transcriptase (RT) genes in thermophilic bacteria. A specific RT from Bacillus caldolyticus was difficult to express, with low soluble yields, suggesting a need for ribonucleoprotein isolation for further study.

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Area of Science:

Molecular Biology
Enzymology
Thermophilic Microbiology

Background:

Reverse transcriptases (RTs) are crucial enzymes involved in RNA-to-DNA synthesis.
Exploring RTs in thermophilic organisms offers potential for novel enzymatic properties and applications.
Group II intron-encoded RTs are found in various prokaryotes and eukaryotes.

Purpose of the Study:

To survey thermophilic bacteria for reverse transcriptase genes.
To isolate and characterize a novel RT from Bacillus caldolyticus.
To investigate expression strategies and activity of the thermophilic RT.

Main Methods:

Degenerate primer PCR targeting known RT sequences.
Genomic walking for gene isolation.
Cloning into Escherichia coli and baculovirus expression vectors.
Assessing RT activity using poly(rC)*p(dG)(12-18) substrate.

Main Results:

Six of 34 thermophilic isolates yielded PCR products indicating RT genes.
A putative RT from Bacillus caldolyticus (EA1) was cloned but showed poor solubility in E. coli.
Expression in a baculovirus system yielded low protein amounts.
Only approximately 10% of the detected RT activity was soluble.

Conclusions:

Thermophilic bacteria harbor diverse reverse transcriptase genes.
Expression and purification of the Bacillus caldolyticus RT present significant challenges.
Further analysis may require isolating the RT as a ribonucleoprotein complex to achieve sufficient quantities.