CSM News Electronic Edition Volume 2, number 21 June 11, 1995 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web through www.nwu.edu. ---------- Abstracts ---------- The Dictyostelium discoideum mitochondrial genome: a primordial system using the universal code and encoding hydrophilic proteins atypical of metazoan mitochondrial DNA. R.A.Cole & K.L.Williams Macquarie University, Sydney, NSQW 2109 Australia Journal of Molecular Evolution, in press. A 3345 bp fragment of Dictyostelium discoideum mitochondrial DNA has been sequenced. This fragment contained the 80 kDa subunit of comlex 1 (NADH;Ubiquinone oxidoreductase), encoding a predicted amino acid sequence of 688 residues and a molecular mass of 79,805 daltons which is nuclear encoded in other metazoa. The C-terminus of the D.discoideum complex 1 gene shared a 10 bp overlap with NADH:Ubiquinone oxidoreductase chain 5 (ND5), while 21 bp 5' were three tRNA genes (two isoleucine and a histidine) and a further 25 bp 5' were three tRNA genes is the partial sequence (104 residues) of an unidentified open reading frame (ORF104). Both the 80 kDa subunit and the ORF104 were hydrophilic and highly charged suggesting they are not membrane associated, unlike most mitochondrially encoded proteins in the metazoa. Sequence analysis of the 80 kDa subunit, its adjacent ND5 gene and ORF104 indicate the universal stop codon TGA, which codes for tryptophan in all non-plant mtDNA, is either unassigned or coding for a stop codon in D.discoideum. This suggests that D. discoideum mitochondrial DNA has not undergone even the first evolutionary change from the universal code. The large size of the mitochondrial genome (54 kb), the lack of intergenic sequence and the apparent use of the universal code suggests D.discoideum mitochondrial DNA may encode many primitive genes which are nuclear encoded in higher organisms. ----------------------------------------------------------------------- Dictyostelium discoideum mitochondrial DNA encodes a NADH:ubiquinone ocidoreductase subunit which is nuclear encoded in other eukaryotes. R.A.Cole, M. B. Slade & K. L. Williams Macquarie University, Sydney, NSW 2109 Australia Journal of Molecular Evolution, in press. Complex I, a key component of the mitochondrial electron transport system, is thought to have evolved from at least two separate enzyme systems prior to the evolution of mitochondria from a bacterial endosymbiont, but the genes for one of the enzyme systems are thought to have subsequently been transferred to the nuclear DNA. We demonstrated that the cellular slime mould Dictyostelium discoideum retains the ancestral characteristic of having mitochondria encoding at least one gene (80 kDa subunit) that is nuclear encoded in other eukaryotes. This is consistent with the cellular slime moulds of the family Dictosteliacae having diverged from other eukaryotes at an early stage prior to the loss of the mitochdonrial gene in the lineage giving rise to plants and animals. The D. discoideum mitochdonrially encoded 80 kDa subunit of complex I exhibits a two-fold higher mutation rate compared with the homologous chromosomal gene in other eukaryotes, making it the most divergent eukaryotic from of this protein. ---------------------------------------------------------------------- DICTYOSTELIUM DISCOIDEUM MUTANTS WITH CONDITIONAL DEFECTS IN PHAGOCYTOSIS Christopher J. Cohen*, Rebecca Bacon*, Margaret Clarke+, Keith Joiner#, and Ira Mellman* Department of Cell Biology* and Section of Infectious Diseases#, Department of Internal Medicine Yale University School of Medicine 333 Cedar Street, PO Box 3333 New Haven, Connecticut 06510 and Program in Molecular and Cell Biology+ Oklahoma Medical Research Foundation Oklahoma City, Oklahoma 73121 THE JOURNAL OF CELL BIOLOGY, in press Abstract We have isolated and characterized Dictyostelium discoideum mutants with conditional defects in phagocytosis. Under suspension conditions, the mutants exhibited dramatic reductions in the uptake of bacteria and polystyrene latex beads. The initial binding of these ligands was unaffected, however, indicating that the defect was not in a plasma membrane receptor. Due to the phagocytosis defect, the mutants were unable to grow when cultured in suspensions of heat-killed bacteria. The mutants exhibited normal capacities for fluid phase endocytosis and grew as rapidly as parental (AX4) cells in axenic medium. Both the defects in phagocytosis and growth on bacteria were corrected when the mutant Dictyostelium cells were cultured on solid substrates. Reversion and genetic complementation analysis suggested that the mutant phenotypes were due to single gene defects. While the precise site of action of the mutations was not established, they are likely to affect an early signaling event since the binding of bacteria to mutant cells in suspension was unable to trigger the localized polymerization of actin filaments required for ingestion; other aspects of actin function appeared normal. This class of conditional phagocytosis mutant should prove to be useful for the expression cloning of the affected gene(s). ------------------------------------------------------------------------ The Ga subunit Ga4 couples to pterin receptors and identifies a novel signaling pathway that is essential for multicellular development in Dictyostelium Jeffrey A. Hadwiger, Susan Lee, and Richard A. Firtel Department of Biology University of California, San Diego La Jolla, California 92093-0634 telephone: 619-534-2788 fax: 619-534-7073 Proceedings of the National Academy of Sciences, in press ABSTRACT In this paper, we shown that the Ga subunit Ga4 couples to pterin receptors and identifies a novel signaling pathway that is essential for multicellular development in Dictyostelium. Ga4 is developmentally regulated, is essential for proper morphogenesis and spore production, and functions cell-non-autonomously. We show that Ga4 is coupled to receptors (aFAR) that activate chemotaxis and adenylyl and guanylyl cyclases in response to folic acid during the early stages of development, and to a late class of folate receptors (bFAR) that have a different specificities for pterins. Ga4 is preferentially expressed in cells randomly distributed within the aggregate that are a component of the anterior-like cell population, and it is not detectably expressed in prespore cells. Our results suggest that an endogenous factor, possibly a pterin, produced during multicellular development is a requisite signal for multicellular development, acting through Ga4. We propose that the Ga4-expressing cells function as a regulatory cell type controlling prespore cell fate, possibly in response to an endogenously produced pterin. Our results indicate that Ga4 and Ga2 have parallel functions in mediating cellular responses to folate (pterins) and cAMP, respectively. -------------------------------------------------------------------- [End CSM News, volume 2, number 21]