CSM News Electronic Edition Volume 6, number 4 February 17, 1996 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== THE VACUOLAR PROTON PUMP OF DICTYOSTELIUM DISCOIDEUM: MOLECULAR CLONING AND ANALYSIS OF THE 100-kDa SUBUNIT Tongyao Liu and Margaret Clarke Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 J. Cell Sci., in press. ABSTRACT The vacuolar proton pump is a highly-conserved multimeric enzyme that catalyzes the translocation of protons across the membranes of eukaryotic cells. Its largest subunit (95-116-kDa) occurs in tissue and organelle-specific isoforms and thus may be involved in targeting the enyzme or modulating its function. In amoebae of Dictyostelium discoideum, proton pumps with a 100-kDa subunit are found in membranes of the contractile vacuole complex, an osmoregulatory organelle. We cloned the cDNA that encodes this 100-kDa protein and found that its sequence predicts a protein 45% identical (68% similar) to the corresponding mammalian proton pump subunit. Like the mammalian protein, the predicted Dictyostelium sequence contains six possible transmembrane domains and a single consensus sequence for N-linked glycosylation. Southern blot analysis detected only a single gene, which was designated vatM. Using genomic DNA and degenerate oligonucleotides based on conserved regions of the protein as primers, we generated products by polymerase chain reaction that included highly variable regions of this protein family. The cloned products were identical in nucleotide sequence to vatM, arguing that Dictyostelium cells contain only a single isoform of this proton pump subunit. Consistent with this interpretation, the amino acid sequences of peptides derived from a protein associated with endosomal membranes (Adessi et al., J. Cell Sci. 108:3331-7, 1995) match the predicted sequence of the protein encoded by vatM. Thus, a single isoform of the 100-kDa proton pump subunit appears to serve in both the contractile vacuole system and the endosomal/lysosomal system of Dictyostelium, arguing that this subunit is not responsible for regulating the differing abundance and function of proton pumps in these two compartments. Gene targeting experiments suggest that this subunit plays important (possibly essential) roles in Dictyostelium cells. ------------------------------------------------------------------- Dictyostelium discoideum contains a family of calmodulin related EF- hand proteins that are developmentally regulated Bernadette Andre$, Angelika A. Noegel* and Michael Schleicher$ $Adolf-Butenandt-Institut/Zellbiologie, Schillerstr. 42, 80336 Muenchen; *Max-Planck-Institut fuer Biochemie, 82152 Martinsried, Fed. Rep. of Germany FEBS Letters, in press Abstract A full length genomic DNA fragment that codes for a novel EF-hand protein from Dictyostelium discoideum was cloned and sequenced. The protein is composed of 168 amino acids and contains four consensus sequences that are typical for Ca2+-binding EF-hand domains. The protein sequence exhibits only minor similarities to other calmodulin-type proteins from Dictyostelium. The genomic DNA harbors two short introns; their positions suggest that the gene is unrelated to the EF-hand proteins from the calmodulin group. Northern blot analysis showed that the mRNA level was significantly increased during development. Polyclonal antibodies raised against the recombinant protein recognized in Western blots a protein of about 20 kDa. Like the mRNA, also the protein was more abundant in developing cells. Overlay experiments with 45Ca2+ indicated that the EF-hands in fact have Ca2+-binding activity. The recent description of CBP1, another calmodulin-type Dictyostelium protein that is upregulated during development (Coukell et al., FEBS-Lett. 362:342-346, 1995), suggests that D. discoideum contains a family of EF-hand proteins that have specific functions during distinct steps of development. We therefore designate the protein described in this report as CBP2. ---------------------------------------------------------------------- Ordered yeast artificial chromosome clones representing the Dictyostelium discoideum genome Adam Kuspa* and William F. Loomis@ * Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030 @Department of Biology, University of California, San Diego, La Jolla , CA 920 Proc. Natl. Acad. Sci., in press. Abstract High resolution gene maps of the 6 chromosomes of Dictyostelium discoideum have been generated by a combination of physical mapping techniques. A set of yeast artificial chromosome clones has been ordered into overlapping arrays that cover >98% of the 34 megabase-pair genome. Clones were grouped and ordered according to the genes they carried, as determined by hybridization analyses with DNA fragments from several hundred genes. Congruence of the gene order within each arrangement of clones with the gene order determined from whole genome restriction site mapping indicates that a high degree of confidence can be placed on the clone map. This clone-based description of the Dictyostelium chromosomes should be useful for the physical mapping and subcloning of new genes, and facilitate more detailed analyses of this genome. -------------------- Table 1. Newly Mapped Loci Locus Product (Phenotype) Chromosome Reference ________________________________________________________ abcA putative transporter 1 a abpD actin binding protein 4 b abpE actin binding protein 3 b act actins - a aldA aldehyde dehydrogenase 4 a aldB aldehyde dehydrogenase 6 a aleA (chemotaxis defective) 2 c arfB ADP ribosylation factor 3 d cabA2 actin/calmodulin bind 6 e cabA3 actin/calmodulin bind 3 e camA1 calmodulin 6 (10) camA2 calmodulin 3 (10) cbpA calcium binding protein 2 (11) cdcE cdc2-related protein 6 (12) cinB (cycloheximide induced) 4 (13) cinC (cycloheximide induced) 6 (13) coaA coactosin 1 (14) comA comitin 5 (15) copB b-COP 4 b corA coronin 1 (16) cudA (culmination defective) 2 f cvmA contractile vac memb. 6 e cycB cyclinB 2 (12) dagG (defective aggregation) 2 a dhkC histidine kinase 4 g dhkD histidine kinase 1 g DIRS8 DIRS subfragment 4 a dlaA dihydrolipoacetyltrans. 3 h fpaA fucose protein 2 (17) fpaB fucose protein 1 (17) ftbA (defective fruits) 2 i fusC gp138B-like 1 c fusD gp138B-like 5 c gerA germ. protein (p109) 4 (18) glpD glycogen phosphorylase 5 (19) gnlA (gnarled fruits) 4 i gppA gp100 6 (20) helC helicase 3 j ksnC kinesin 3 6 k ksnF kinesin 6 1 k ksnG kinesin 7 3 k lmcB (lack of multicell) 3 g mapA microtubule associated 2 l mdrA putative transporter 6 a migA (migration defective) 4 a mlcR myosin light chain 4 (21) mndJ (blocked at mounds) 4 j motA (chemotaxis defective) 1 h mrpA putative transporter 4 m mrpB putative transporter 6 m mrpC putative transporter 2 a natA N-term acetyltransfer 2 h ncsB non-cell specific cDNA 4 (22) ndhD NADH dehydrogenase 1 a nosA (no spores) 6 f pabA polyA binding protein 6 a pagB (preaggregation) 2 g pagC (preaggregation) 4 g papA poly-A polymerase 2 a patA P-type ATPase 3 n pkbA protein kinase K2.6 1 o pkfC protein kinase DK1 4 p pmpA putative membrane 1 q ponA ponticulin 1 (23) proB profilin II 4 (24) prtA proteosomal a-sub M3 6 (25) prtB proteosomal a-sub 7-1 2 (25) pspC prespore gene 2 r pspC prespore gene 2 r pspE prespore gene 1I 4 (22) pspF prespore gene 2H 3 (22) pspG prespore gene 3F 2 (22) pspH prespore gene 6C 3 (22) pspJ prespore gene 7F 2 (22) pspL prespore gene 1H 2 (22) pstA prestalk gene 5G 4 (22) ptpC phosphotyr. pase 3 2 (26) ptpE phosphotyr. pase 3 1 (26) purA adenylosuccinate synth 4 b rasG ras homolog (growth) 1 (27) repE DNA repair gene homolog 4 s rioT putative transcripfactor5 t rpgB ribosomal protein 3 (28) rpgF ribosomal protein 6 u rtoA (altered ratios) 2 i rtoB (altered ratios) 2 i schA (signaling defective) 2 c sfbA (small fruiting bodies) 1 (4) smlA (small fruiting bodies) 4 i snrU U1 protein 1 a sokA (stringy on Klebsiella) 2 p sqpA serine/glutamine-rich 3 v talA talin 5 (29) timA (tipped mound arrest) 1 a tubA alpha tubulin 6 (30) ubcA ubex52 3 (31) ubpA (defective aggregation) 4 i ubqD ubiquitin 1 (32) ubqE ubiquitin 1 (32) ubqF ubiquitin 4 (32) uceA ubiquitin conjugating 3 w uppA UDPG pyrophosphorylase 1 (33) vagA (signaling defective) 2 c vatP vacuolar ATPase 2 n vseC vegetative expression 1 (28) vseE vegetative expression 1 (28) vsgA putative vsvG homolog 6 a vsgB putative vsvG homolog 5 a yelA (yellow mounds) 3 a _________________________________________________________________ Lettered references indicate the source of unpublished probes for the genes listed: a; This Work, b; A. Noegel, c; P. Devreotes, d; C. Weijer, e; M. Clarke, f; R. Kessin, g; C. Singleton, h; G. Gerisch, i; R. Gomer, j; R. Kay, k; R. Vale, l; M. Kimble, m; T. Wang, n; B. Coukell, o; M. Veron, p; J. Williams, q; W. Nellen, r; D. Hames, s; S. Alexander, t; S. Bozarro, u; H. Ennis, v; G. Podgorski, and w; E. Luna. -------------------------------------------------------------------- [End CSM-News, volume 6, number 4]