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 can also be used to construct double and triple-knockout strains. 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.

The frequency of transformation is high enough to allow the complementation of mutants created by chemical mutagenesis. The method has been demonstrated using a genomic library to complement null mutants in several previously identified genes, but this was an isolated success until recently. In unpublished work, a cDNA library has been used to complement a cytokinesis mutant. Not only was the original mutated gene identified, but several multi-copy suppressors were also obtained in the same experiment, allowing a pathway to be explored (1).

The sequencing of the D. discoideum genome is well underway through a coordinated international effort.The haploid genome is 3.4 x 10⁷ base pairs (34 Mbp) consisting of six chromosomes of 4 to 7 Mbp. Introns are relatively rare and promoters and introns are relatively short. The introns and promoters are usually more than 90% AT, distinguishing them from the GC-rich coding regions of the genes. The number of genes lies between the 6000 of S. cerevisiae and the 19000 of C. elegans.  There are likely to be several thousand genes that have homologues in higher eukaryotes and that are absent in S. cerevisiae. The AT bias facilitates prediction of  these ORFs and these novel genes can be analyzed with powerful molecular genetics.

Several sequencing projects are well coordinated.  A project in Japan has completely sequenced 5560 non-redundant cDNAs expressed at the multicellular stages of development and is now sequencing an equivalent number from aggregating and growing cells. Genomic DNA sequencing has been funded from the DFG (A. Noegel, Cologne, and A. Rosenthal, Jena, Germany); the National Institutes of Health (A. Kuspa and R. Gibbs, Baylor College, TX, USA); and the European Union (Sanger Centre, Pasteur Institute, and Cologne University in a consortium led by B. Barrell). The strategy is to sequence individual chromosomes separated by pulsed field gels, an approach that avoids cloning with its attendant dangers of chimerism, deletion and rearrangement and simplifies the process of dividing the genome among the sequencing laboratories. Each chromosome will be shotgun sequenced to about a five-fold redundancy with assembly of this sequence expected to produce many contigs. These will be assembled into larger contigs by matching to existing mapped yeast artificial chromosomes of the same chromosome and to higher resolution maps now under construction. A shotgun sequencing of the entire genome has been performed at Jena to provide a preview of the gene content of D. discoideum. Currently 70 Mbp of DNA sequence can be searched at the Jena Web Site.  Many genes with homologues in higher eukaryotes have been identified by both the cDNA and the genome sequencing projects.