A faster way to generate hypomorphic mutations

Researchers have developed a simple and rapid method for generating partial loss-of-function mutations in a wide variety of organisms.

The standard approach for elucidating the function of a novel gene is through the phenotypic analysis of mutations introduced into that gene. While null mutations can be very informative, the lethality of null mutants of essential genes prevents the analysis of those genes’ functions. This makes partial loss of function (i.e., hypomorphic) mutations valuable since they allow cells or organisms to survive to later stages of development, when they can be examined for changes in phenotype.

Obtaining hypomorphic mutations using standard forward genetic screens is time-consuming and laborious. Now, researchers led by Sergej Djuranovic at the Washington University School of Medicine have developed a method to quickly and easily generate a series of hypomorphic mutations expressing a range of reduced levels of the protein of interest.

The new method is based on the use of a novel cis translational regulatory element, previously identified by the team, that exists in most eukaryotes. The researchers found that poly(A) tracts—stretches of consecutive adenines that encode polylysine runs—in the open reading frames of certain eukaryotic genes cause ribosome stalling and frameshifts during translation, resulting in reduced protein production and degradation of the mRNA.

Djuranovic and his colleagues reasoned that inserting poly(A) tracts of various lengths into a gene of interest might systematically generate a series of hypomorphic mutations in that gene. When they inserted increasingly longer poly(A) sequences into a gene, they saw decreasing levels of protein expression from the gene, independent of promoter strength. This method works across a broad range of experimental models, including E. coli, protozoans, plant tissues, fruit flies, and human cell lines, and will greatly facilitate the genetic analysis of gene function.