Isolation and characterization of Saccharomyces cerevisiae mutants defective in chromosome transmission in an undergraduate genetics research course.

An upper-level genetics research course was developed to expose undergraduates to investigative science. Students are immersed in a research project with the ultimate goal of identifying proteins important for chromosome transmission in mitosis. After mutagenizing yeast Saccharomyces cerevisiae cells, students implement a genetic screen that allows for visual detection of mutants with an increased loss of an ADE2-marked yeast artificial chromosome (YAC). Students then genetically characterize the mutants and begin efforts to identify the defective genes in these mutants. While engaged in this research project, students practice a variety of technical skills in both classical and molecular genetics. Furthermore, students learn to collaborate and gain experience in sharing scientific findings with others in the form of written papers, poster presentations, and oral presentations. Previous students indicated that, relative to a traditional laboratory course, this research course improved their understanding of scientific concepts and technical skills and helped them make connections between concepts. Moreover, this course allowed students to experience scientific inquiry and was influential for students as they considered future endeavors.

Subtractive immunization: a tool for the generation of discriminatory antibodies to proteins of similar sequence.

Antibodies specific for a protein of interest are invaluable tools for monitoring the protein's structure, location and activity. Due to the tendency of an immune system to mount a response toward the abundant, immunodominant epitopes in a protein mixture, difficulties are inherent in the isolation of antibodies specific for proteins that are rare or poorly immunogenic. Likewise, isolation of antibodies specific for a protein with significant sequence similarity to other proteins, such as those derived from protein engineering, may be challenging. Subtractive immunization is a technique proven to facilitate efforts to produce monoclonal antibodies specific for antigens that are present in low abundance in a protein mixture, poorly immunogenic and/or similar in sequence or structure to other proteins. This protocol provides a detailed, stepwise procedure for the isolation of antibodies specific for a protein with sequence similarity to other proteins. As an example, we describe methods established to isolate antibodies specific to a methionine-enriched variant of soybean vegetative storage protein beta (VSPbeta-Met) that shares 91.8% amino acid sequence identity to the wild-type protein (VSPbeta-WT). These methods include cyclophosphamide-induced immunosuppression of mice for the wild-type protein followed by immunization with VSPbeta-Met. As a result of this procedure, mouse polyclonal antibodies that exhibited 10-fold greater reactivity with VSPbeta-Met than VSPbeta-WT in an ELISA were generated. It is anticipated that this strategy will have utility for generating antibodies specific to protein variants derived from protein engineering.