Although it was previously reported that Dictyostelium discoideum possessed a single ras gene (Ddras) that was maximally expressed during the pseudoplasmodial stage of development, a second ras gene (DdrasG), has been isolated and characterized. It encodes a protein that is similar to the protein encoded by Ddras and the human ras proteins. However, in contrast to Ddras, the DdrasG gene was only expressed during growth and early development. The two ras proteins may fulfill different functions: the DdrasG protein having a role during cell growth and the Ddras protein having a role in signal transduction during multicellular development. However, the expression of the DdrasG gene throughout development did not appear to have a detrimental effect on differentiation. Although other eukaryotic organisms possess more than one ras gene, D. discoideum is thus far unique in expressing different ras genes at different stages of development. Ras genes are members of a large ras-related multigene family that has been found in a wide variety of organisms. A ras-related gene was isolated from D. discoideum that hybridized to both the Ddras and DdrasG genes under low, but not under high stringency conditions. The predicted amino acid sequence shows a high degree of sequence identity with the human rap proteins and thus has been designated Ddrapl. During vegetative and early development a single 1.1 kb mRNA was present, but by aggregation this transcript was no longer detected and two new transcripts of 1.0 and 1.3 kb were observed and were present throughout the remainder of development. The maximum levels of the Ddrapl specific mRNAs appeared during aggregation and culmination, developmental stages where the levels of DdrasG and Ddras messages were declining. The reciprocal nature of the Ddrapl gene expression with respect to that of the two ras genes suggests the possibility that the ras and rap gene products in D. discoideum have antagonistic roles. Antibodies that are specific for the Ddras, DdrasG and Ddrapl proteins have been generated and can be used to help elucidate the biological functions of the individual proteins. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Indiana University-Purdue University Indianapolis (IUPUI) / The lipid profile of Dictyostelium discoideum, a cellular slime mold found evolutionarily between plants and animals, has never been clearly defined. To address this, the fatty acid content of vegetative cells was analyzed by gas chromatography-mass spectrometry of fatty acid methyl esters and their identities verified with synthesized authentic standards. The synthetic scheme developed to produce the unusual fatty acids found in D. discoideum was engineered to afford the labeling of compounds (2H) for use in feeding studies to elucidate the fatty acid elongation and desaturation pathways present in D. discoideum. After establishing the fatty acid profile and acyl metabolic pathway, an initial understanding the complex lipids present in D. discoideum, chiefly sphingolipids, was sought. Triple quadrupole and quadrupole time-of flight mass spectrometers equipped with electrospray ionization sources were used to identify these complex lipids.