Dicty News Electronic Edition Volume 11, number 11 December 5, 1998 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" ============= Abstracts ============= Expression of Chicken Gizzard RLC Complements the Cytokinesis and Developmental Defects of Dictyostelium RLC Null Cells Pengxin Chen, Bernard M. Chaudoir, Kathleen M. Trybusl,2, and Rex L. Chisholm* Department of Cell and Molecular Biology and Robert H. Lurie Cancer Center, Northwestern University Medical School 303 E. Chicago Avenue 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University Waltham, MA 02254, 2 Current Address: Dept. of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405 J. Muscle Res. Cell Motil, in press Abstract Dictyostelium RLC null cells have defects in cytokinesis and development that can be rescued by expression of either the wild type Dictyostelium RLC or an RLC mutant that cannot be phosphorylated by MLCK (S13A) (Ostrow et al., 1994). The wild type and S13A mutant LCs rescued the cells equally well, despite the fact that RLC phosphorylation increases purified Dictyostelium myosin's activity 5-fold. In this report we assess the ability of foreign RLCs to rescue the RLC null phenotype. The RLC from smooth muscle myosin, whose activity is tightly controlled by phosphorylation, rescued the null cell phenotype. The purified hybrid myosin had activity and motility comparable to phosphorylated Dictyostelium myosin. In contrast, cells expressing skeletal muscle RLC were deficient in cytokinesis and development, despite having an activity and motility similar to that of myosin with the unphosphorylatable S13A mutant RLC. Neither foreign LC was phosphorylated when expressed in Dictyostelium. These results suggest that the level of actin-activated ATPase activity and motility is not the sole determinant of proper myosin function in vivo. Other heavy chain/light chain interactions, which occur only with the native RLC and smooth muscle RLC, appear to be necessary for optimal function. ---------------------------------------------------------------------------- The mitochondrial adenine nucleotide translocator from Dictyostelium discoideum. Functional characterisation and DNA sequencing. Mireille Bof, Gerard Brandolin, Michel Satre and Gerard Klein Laboratoire de Biochimie et Biophysique des Systemes Integres (UMR 314 CEA-CNRS), CEA-Grenoble, France. Eur. J. Biochem., in press SUMMARY The mitochondrial adenine nucleotide translocator (ANT) catalyses the exchange of ATP and ADP between the mitochondria and the cytosol. We have cloned and sequenced the gene encoding the Dictyostelium discoideum adenine nucleotide translocator protein (DdANT) and analysed its transcriptional regulation. The single copy D. discoideum ant gene encodes a protein of 309 amino acid residues with a predicted molecular mass of 33469 Da and a pI of 9.85. These values are comparable to those of ANTs from mammals, insects or fungi. The long N-terminal extension characteristic of plant ANT is absent in DdANT. The protein coding region of the D. discoideum ant gene is interrupted by three introns. Polyclonal antibodies directed against the beef heart mitochondrial ANT or its C-terminal peptide recognised the D. discoideum protein. Northern analysis revealed that the expression of the D. discoideum ant gene decreased rapidly during first hours of multicellular development but the amount of protein remained stable throughout differentiation. ---------------------------------------------------------------------------- Dgp1 and Dfp1 are Closely Related Plasmids in the Dictyostelium Ddp2 Plasmid Family CHAD M. GONZALES, TRINA D. SPENCER, SCOTT S. PENDLEY, AND DENNIS L. WELKER1 Program in Molecular Biology, Department of Biology, Utah State University, Logan, Utah, 84322-5305 PLASMID, in press. Summary: Dictyostelium plasmids Dgp1 and Dfp1, two members of the Ddp2 plasmid family, are 86 % identical in nucleotide sequence. These small (4481 and 5015 bp), high copy number, nuclear plasmids both carry a gene homologous to the Ddp2 rep gene and a long 0.47 to 0.48 kb inverted repeat region. Their Rep proteins are 82.8% identical in amino acid sequence and carry all ten of the conserved peptide sequence motifs found in the Ddp2 family Rep proteins. Unlike other members of this family, Dgp1 carries two copies and Dfp1 carries four copies of a 162 to 166 bp direct repeat element. Both the direct and inverted repeat elements, as well as the promoter of the rep gene, are highly conserved (81 to 90% identical) between Dgp1 and Dfp1. In contrast, these regions are not highly conserved and the Rep proteins are only about 40% identical among the other known members of the plasmid family. ---------------------------------------------------------------------------- Cloning of a cDNA encoding a new calcium-binding protein from Dictyostelium discoideum and its developmental regulation Young-Hoon Han, Sa-Ouk Kang* Laboratory of Biophysics, Department of Microbiology, College of Natural Sciences and Research Center for Molecular Microbiology, Seoul National University, Seoul 151-742, Republic of Korea FEBS Lett., in press. Abstract: By employing 2D-PAGE, a small protein differentially expressed during the development of Dictyostelium discoideum was discovered. The full cDNA of this protein was cloned using RT-PCR technique. The deduced protein is composed of 166 amino acids containing four EF-hand domains typical for calcium-binding proteins and was named CBP3. This protein shows little amino acid sequence homology with other calcium-binding proteins from Dictyostelium except EF-hand domains. The CBP3 mRNA was absent in vegetative amoebae and accumulated maximally at 6 h of the development on filters. The mRNA level decreased thereafter and disappeared after 12 h of the development, while the protein level peaked at 8 h of the development and was maintained constant thereafter. The mobility of CBP3 on SDS gel was shifted by the treatment of EGTA, confirming the Ca2+-binding activity of the protein. ---------------------------------------------------------------------------- Involvement of the Ca2+ ATPase PAT1 and the Contractile Vacuole in Calcium Regulation in Dictyostelium discoideum John Moniakis, Barrie Coukell and Agnes Janiec Department of Biology, York University, 4700 Keele St., Toronto, Ontario, CANADA J. Cell Sci. in press Abstract In Dictyostelium discoideum, the Ca2+ ATPase, PAT1, is localized to membranes of the contractile vacuole and its expression is up-regulated substantially when the cells are grown in Ca2+-rich media. In this study, we have analyzed the cellular/molecular mechanisms regulating PAT1 expression and examined the role of PAT1 and the contractile vacuole in Ca2+ regulation. During both growth and development, Dictyostelium cells respond to low millimolar concentrations of extracellular Ca2+ and up-regulate PAT1 in a few hours. This process is dependent on protein synthesis and the serine/threonine phosphatase, calcineurin. Immunofluorescence analysis indicates that the up-regulated PAT1 is associated mainly with the contractile vacuole, but it is also on the plasma membrane. This latter finding suggests that the contractile vacuole fuses with the plasma membrane to eliminate excess intracellular Ca2+. In support of this idea, it was observed that conditions which impair contractile vacuolar function reduce the rate of Ca2+ secretion. It was also found that cells deficient in PAT1, due to the expression of antisense patA RNA or to the presence of calcineurin antagonists, grow normally in low Ca2+ medium but poorly or not at all in high Ca2+. Together, these results suggest that PAT1 and the contractile vacuole are components of a Ca2+ sequestration and excretion pathway which functions to help maintain Ca2+ homeostasis, especially under conditions of Ca2+ stress. ---------------------------------------------------------------------------- The Dictyostelium Developmental cDNA Project: Generation and Analysis of Expressed Sequence Tags from the First-Finger Stage of Development Takahiro Morio(1), Hideko Urushihara(1), Tamao Saito(2), Yoshihiro Ugawa(3), Hideaki Mizuno(1), Motonobu Yoshida(4), Ryuji Yoshino(1), Biswa Nath Mitra(1), Min Pi(1), Tomihiro Sato(1), Keiko Takemoto(5), Hiroo Yasukawa(6), Jeffrey Williams(7), Mineko Maeda(8), Ikuo Takeuchi(9), Hiroshi Ochiai(2), and Yoshimasa Tanaka(1,*) (1)Institute of Biological Sciences, Univ. Tsukuba, Japan, (2) Division of Biological Science, Graduate School of Sciences, Hokkaido Univ., Japan, (3) DNA Information & Stock Center, National Institute of Agrobiological Resources, Japan, (4) Research Institute of Food Science, Kinki Univ., Japan, (5) Institute for Virus Research, Kyoto Univ., Japan, (6) Faculty of Engineering, Toyama Univ., Japan, (7) Department of Anatomy and Physiology, University of Dundee, UK, (8) Department of Biology, Graduate School of Science, Osaka Univ., Japan, (9) Novartis Foundation for the Promotion of Science, Japan DNA Research, in press Abstract In an effort to identify and characterize genes expressed during multicellular development in Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNAs from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts. The other, library L, which consists of 8448 clones, has longer inserts. We have sequenced all the selected clones in the library S from their 3’-ends and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs derive from known Dictyostelium genes and 910 are suggested to have significant similarity to genes of Dictyostelium or of other organisms. From the library L, 1132 clones were randomly sequenced and this generated 471 non-redundant ESTs. In combination, the ESTs from the two libraries will represent 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism. ---------------------------------------------------------------------------- [End Dicty News, volume 11, number 11]