Cold-sensitive phenotypes have helped us understand macromolecular assembly and biological phenomena, yet few attempts have been made to understand the basis of cold sensitivity or to elicit it by design. We report a method for rational design of cold-sensitive phenotypes. The method involves generation of partial loss-of-function mutants, at either buried or functional sites, coupled with selective overexpression strategies. The only essential input is amino acid sequence, although available structural information can be used as well. The method has been used to elicit cold-sensitive mutants of a variety of proteins, both monomeric and dimeric, and in multiple organisms, namely Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster. This simple, yet effective technique of inducing cold sensitivity eliminates the need for complex mutations and provides a plausible molecular mechanism for eliciting cold-sensitive phenotypes.conditional mutants | rational design | cold sensitivity | heat-induced expression | transfer between organisms C onditional mutants are powerful tools for studying gene function in vivo. A conditional mutant retains the function of a gene under one set of conditions, called permissive, and lacks that function under a different set of conditions, called restrictive, whereas the wild type (WT) phenotype is similar across both conditions. Cold-sensitive (cs) mutants behave like loss-of-function mutants at temperatures lower than a cutoff temperature, but have WT-like phenotypes at higher temperatures. In contrast, temperature-sensitive (ts) mutants show heat sensitivity and behave like the WT below the restrictive temperature. Both cs and ts mutants can provide important information about protein structure, function, and assembly. cs mutants are rarer than ts mutants, and the molecular basis for generation of cs phenotypes is currently unclear.cs mutants have been used to analyze various biological phenomena, most commonly the cell cycle (1-3), ribosome assembly (4-8), and protein export (9-11), as well as to understand macromolecular structure-function relationships (12, 13). Various ts and cs variants also have helped us understand P22 phage coat protein assembly. Using a combination of both ts and cs mutants causing defects at different stages of the assembly pathway, the order of the various steps occurring along this pathway has been determined (14). Thus, these conditional mutants have led to a greater understanding of phage genetics, protein folding, and macromolecular assembly (15)(16)(17). Similarly, cs and ts mutants of different genes essential for cell division, in combination with temperature-shift experiments, have been used to understand the cell cycle phases in which each of these genes act (18).Various changes at the amino acid level or gene regulation level can cause cold sensitivity. Some cs mutants have been suggested to result from altered feedback inhibition of certain metabolic pathways at lower temperatures (19,20). Cold sensitivity also has been attribut...

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