Molecular Genetics

Most of the molecular genetic techniques typically associated with S. cerevisiae are available in D. discoideum. Random mutagenesis is performed with high efficiency extrachromosomal vectors. Some of the most elegant studies have correlated the behavior of wild type and mutated myosin heavy chains in single molecule assays with functions in cytokinesis in living cells.  For example myosins with point mutations isolated by phenotypic screens can be purified in milligram quantities and assessed in in vitro assays.  Similar random mutagenesis studies have explored the function of surface receptors, G-proteins, and adenylyl cyclases.

Non-essential genes are easily disrupted by homologous recombination. So far, over 400 genes involved in cell motility, signal transduction, and cell differentiation have been targeted. Strains with multiple gene deletions are constructed by consecutive transformations using different selectable markers. Parasexual genetics and the cre-lox system (Faix et al., 2004) can also be used to construct double and triple-knockout strains. many of those mutants are available at the Dicty Stock Center. Because the organism displays most of its interesting behavior as either a haploid or a diploid, recessive phenotypes are immediately apparent and are often detectable by clonal morphology. Since the cells are free living, gene deletions that might be lethal in other organisms can often be productively studied in D. discoideum.

Restriction Enzyme-Mediated Integration REMI is used to create libraries of strains containing random genomic insertions. Cells are electroporated with a mixture of a linearized plasmid and a restriction enzyme that catalyzes insertion of the plasmid into the genome at corresponding restriction sites. Generally, the transformed cells contain a single copy of the plasmid and the sequences flanking the insertion site can be obtained by rescue in E coli. To verify that the recovered DNA sequence is responsible for the phenotype, the rescued plasmid is used to recreate the genotype by homologous recombination. The frequency of insertion is high enough to also allow suppressor genetics. A large number of new gene products involved in cytokinesis, motility, aggregation, and later development have been isolated. Many REMI mutants in a wild-type background and corresponding genes are available from the Developmental Gene Project at UCSD and the Baylor College of Medicine (you can view availble mutants here).