A gene is a fundamental unit of heredity, a segment of DNA that carries the instructions for building and maintaining an organism. Sometimes, changes occur in this DNA sequence, known as mutations, which can alter the gene’s instructions. While many mutations lead to obvious changes in an organism’s traits, some are more subtle, revealing their effects only under specific environmental conditions. These are known as conditional mutants.
Temperature is a common and useful environmental condition that scientists manipulate to study these conditional mutants. By simply adjusting the temperature, researchers can control when a gene’s altered function becomes apparent or when it behaves normally. This ability to “switch” a gene’s activity on or off by changing the environment provides a powerful tool for understanding biological processes.
The Mechanism of Temperature Sensitivity
Temperature-sensitive mutants function based on how temperature influences the stability of the proteins produced by their altered genes. At a “permissive temperature,” the mutant protein can fold correctly and perform its normal biological task, allowing the organism to grow without apparent issues. This temperature is typically lower than an organism’s optimal growth temperature, such as 25°C for many laboratory yeasts.
However, when the temperature is raised to a “restrictive temperature,” the subtle change in the protein’s amino acid sequence, caused by the mutation, becomes significant. This alteration makes the protein unstable, leading it to misfold or lose its proper shape, which then renders it non-functional. For instance, a yeast mutant might grow normally at 25°C but fail to grow at a restrictive temperature like 36°C or 37°C because a particular protein loses its function.
Applications in Scientific Research
Temperature-sensitive mutants are invaluable tools in biological research, especially for investigating the roles of genes that are necessary for an organism’s survival. Researchers face a challenge when studying such “essential genes”: simply deleting them would result in the organism’s death, preventing any observation of the gene’s function. This problem is circumvented by using temperature-sensitive variants.
Scientists can grow organisms with temperature-sensitive mutations at the permissive temperature, where the essential gene functions, allowing the organism to survive and develop normally. Then, by shifting the organism to the restrictive temperature, the function of the specific gene is effectively “turned off” at a precise time. This controlled inactivation allows researchers to observe the downstream effects and identify the gene’s role in various cellular processes, such as cell division or protein synthesis.
A notable application involves studying the cell cycle, the series of events cells undergo as they grow and divide. By isolating mutants that arrest at specific stages of cell division when shifted to a restrictive temperature, scientists identified and characterized many genes regulating this complex process. This work, particularly in yeast, was foundational, demonstrating the impact of these mutants on understanding biological mechanisms.
Creating and Identifying Mutants
Creating temperature-sensitive mutants begins by inducing random mutations across a large population of organisms. Scientists often use chemical mutagens, such as ethyl methanesulfonate (EMS), or ultraviolet (UV) radiation, to introduce changes in the DNA sequence. This generates a diverse collection of mutated organisms, increasing the likelihood of finding temperature-sensitive changes.
Once mutations are induced, a screening process is employed to identify the desired temperature-sensitive variants. A common and effective method is replica plating, a technique developed by Joshua and Esther Lederberg. In this procedure, colonies of the mutated organisms are first grown on a “master plate” containing a complete growth medium at the permissive temperature.
A sterile velvet pad or similar transfer tool is pressed onto the master plate, picking up cells from each colony while maintaining their original spatial arrangement. This velvet is then used to “replica plate” these colonies onto two or more new plates. One “daughter plate” is incubated at the permissive temperature (e.g., 25°C), while another is incubated at the restrictive temperature (e.g., 37°C). Colonies that grow on the permissive plate but fail to grow or show significantly reduced growth on the restrictive plate are identified as potential temperature-sensitive mutants.