“Research is to see what everybody has seen and think what nobody has thought.
– Albert Szent-Györgyi [Nobel Prize in Physiology/ Medicine, 1937]
Genetic research is at a colossal high today, and although we know a lot about our genes, the roles of more than 30% of the functional genes in the human body are not really understood. This number can be even lower for other members of the biotic world. Studies to determine gene function involve combinations of various experimental methods at biochemical, cellular, and organismal levels. One such method, that is popularly employed, uses temperature-sensitive mutant genes that behave differently at different temperatures. The process of identifying and generating mutated genes, however, is laborious, time-consuming and relies heavily on chance. It is at this juncture that Prof. Raghavan Varadarajan and his team from the Molecular Biophysics Unit, Indian Institute of Science, Bangalore, suggest an innovative, yet fairly straightforward, technique to study gene functionality, which would make one wonder how no one thought of this earlier!
Most of the ‘functional’ genes in our body function by serving as codes for the machinery inside our cells to produce specific proteins, each of which, in turn, have their own biological roles. Simply put, the function of most genes is the function of the protein that it codes for. So, how would one go about figuring out the role of a particular gene, especially when there is minimal knowledge of the encoded protein product? For organisms that do not maintain a constant body temperature, the use of temperature-sensitive mutations is a classical approach, where combinations of heat and cold-sensitive mutant genes are employed to decipher the role of various genes at different stages of an organism’s life.
The strategy generally involves mutating a particular gene such that its protein product can function normally at regular temperatures, but less efficiently at higher or lower temperatures, depending on the technique employed. For instance, if, bacterial cells — whose original ‘gene of unknown function’ is replaced with a mutated heat-sensitive copy of the same gene — can divide at regular temperatures but not at a higher temperature, one could conclude that the mutated gene plays a major role in bacterial cell division. In contrast, to heat-sensitive mutants, the molecular basis for cold-sensitive mutants is poorly understood and there exists no fool-proof method of successfully generating cold-sensitive mutants for a gene of interest. Hence these are often difficult to isolate. Now, for the first time, Prof. Varadarajan and his team in collaboration with Prof K VijayRaghavan, have devised a straightforward method to generate cold-sensitive mutants of any gene that one might wish to study. Their procedure utilises computer programs to predict the regions of a particular gene that could be mutated to yield a protein product that is destabilized relative to the wild-type (unmutated) gene. Ideally, this ‘unstable protein’ would be less active at all temperatures when compared to the original, non-mutated one, but not completely inactive. After confirming the computer-generated predictions using live model organisms, the mutated gene is transferred into the test organism under the control of a heat-inducible promoter (HIP) that regulates protein production and can be controlled using temperature. When partnered with the mutated gene, these special promoters result in higher production of the protein at high temperatures and lower production at cold temperatures.
At high temperatures, the researchers observed that HIP orchestrated high protein production from the mutated gene. Even though the protein generated was unstable, the test organism could still function normally. However, at low temperatures, there is significantly less mutant protein produced due to the suboptimal functionality of HIP. Here, the organism displays a striking malfunction with respect to some observable trait, providing the researchers with a clue to the gene’s function. Using this method, the researchers successfully established cold-sensitive mutant strains of bacteria and yeast and could also transfer the mutant gene from yeast to fruit flies and still get it to function as desired, in a cold-sensitive fashion.
This study, apart from providing a useful tool in the lab, also offers a plausible explanation for how certain proteins in the cell purport as being cold sensitive. “It’s not that the protein has magically become inactive at lower temperatures”, explains Prof. Varadarajan, “it’s just that, at cold temperatures, the lower expression combined with the intrinsic instability of the mutant leads to an insufficient amount of functional protein.” Although researchers have previously established cold-sensitive mutants in their labs, a molecular rationale of this behaviour was previously lacking.
It is straightforward to generate mutants with lowered activity and HIP’s are also commonly used. However, combining these known concepts to generate cold-sensitive mutants is novel and can ultimately serve as an extremely valuable tool in the elucidation of gene function.