CSM News Electronic Edition Volume 7, number 9 September 28, 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" ============================= Another Sequencing Proposal ============================= Large scale sequence analysis of developmentally regulated mRNAs We all accept the necessity of establishing the sequence of the Dictyostelium genome as rapidly as possible and in a recent CSM newsletter there were two proposals directed towards acheiving this aim; one to sequence the entire genome the other to sequence large numbers of proteins. Here we will describe a large scale sequencing strategy, that we are actively pursuing, which is based upon sequencing cDNA clones. We will explain why we have taken this route but wish to stress that the cDNA strategy should not be viewed as a competitor to direct genomic sequence analysis. In the longer term, when sufficient funds become available, it is imperative that the entire genomic sequence be determined. Rather, the cDNA strategy is something that can be started now, with resources that are presently available and which is entirely complementary to the whole genome sequencing project; the cDNA sequences will be essential if the organisation of the genes identified by genomic sequencing is to be correctly deduced and the genomic sequence will be necessary to ensure that all genes are eventually sequenced. Just as in genome projects in other organisms, such as Drosophila and man, sequence information obtained by the genomic and the cDNA routes will be freely interchanged and we will all reap the benefits of knowing the entire sequence that much more quickly. We aim to sequence between 10,000 and 20,00 cDNA clones derived from mRNA isolated from Dictyostelium slug cells. We estimate that up to one half of the genes expressed at this stage will be sequenced in such a program. It is, therefore, a rapid and relatively inexpensive way of determining the coding potential of a significant part of the Dictyostelium genome. Also, once the sequences of their component cDNA clones are established, the libraries themselves will provide a resource that will greatly facilitate Dictyostelium research. This project has thus far been funded by a Japanese agency, the grant this year being $273,000 with a significant increase expected for 1997. However, to sequence such a large number of clones it will be necessary to obtain collaborators who have grant monies available and who can share the sequencing load. Why should they do this? We believe that sequencing technology has now improved to the point where large-scale sequence analysis is a highly efficient way of studying the function of Dictyostelium genes. It is readily possible to perform homologous gene disruption using cDNA clones and so participants will be able to perform functional analysis on selected cDNAs. This will, in a way, be the converse of REMI and will complement it nicely. We will first identify genes containing domains which suggest that they are likely to have an important function and will then determine the consequences of their disruption. One difference from REMI is that one will know, a priori, what kind of gene one is dealing with so that the frustrations of working with a "pioneer gene" will be removed. Why sequence cDNAs? Determination of the sequence of a large number of cDNA clones has several major attractions. 1) By sequencing 10-20,000 cDNA clones a large fraction of the genomic coding potential will be determined. The genome size, the analysis of gene structures and RNA hybridisation analysis combine to show that there are between 5,000 and 10,000 Dictyostelium genes. About three quarters are expressed during growth and another quarter are activated during development. These are not mutually exclusive populations, so that establishing the sequences of the mRNAs present in the slug will also yield the sequences of some of the genes that are expressed during growth. Admittedly, this will only be a fraction of the vegetative mRNAs but the compelling argument in favour of initially targetting developmental mRNAs is that many of the most interesting facets of the biology of the organism are manifest only during development. 2) The cDNA clones can be categorised by nucleic acid hybridisation. The fact that each cDNA clone derives from a single transcription unit and can be rapidly screened by nucleic acid hybridistaion has great advantages, both in terms of distributing the effort of sequencing and in the speed at which useful information will accrue. The libraries will be gridded at high density and the cDNA clones will be sequentially screened by nucleic acid hybridisation using various probes. We will identify those cDNAs that are prestalk-specific, those that are prespore-specific and those that are DIF inducible. Thus, for every single cDNA clone within the library a gene expression profile will be built up. In this way the cDNA libraries can be sub-divided so that the work of sequencing them can be shared between particular "interest groups". It will also be readily possible to screen for particular conserved sequence domains by hybridisation, again allowing clones from the libraries to be allocated to particular interest groups. 3) Once sequenced the cDNA clones will themselves be an invaluable resource. The cDNA clones and DNA prepared from them will be stored. Once the entire sequence of each clone is known its sequence will be placed in the DictyDB database and also in the generally available sequence banks. Individuals who have a segment of DNA sequence, or reverse translated protein sequence, that aligns with a clone in the library will be sent that clone. This will enormously facilitate research within the field. At the moment many genes are first isolated as genomic fragments, by insertional gene disruption or by PCR, and the cognate cDNAs must then be cloned and sequenced. Repeated many times in different labs around the world, this amounts to a large effort. This project would obviate much of this: it would merely be necessary to obtain a tag sequence, scan the database for the desired clone and then request it. It will also be possible to conduct "computer-cloning" by searching for desired genes based on sequence conservation of particular protein domains. A call for collaborators The speed at which we can procede with this scheme will soon become dependent upon the number of groups who will share with us the work of sequencing and we have appended a more detailed project description for those who are potentially interested in participating. Current Participants: Ikuo Takeuchi (Okazaki) Yoshimasa Tanaka (Tsukuba) Hideko Urushihara (Tsukuba) Takahiro Morio (Tsukuba) Yoshihiro Ogawa(Tsukuba) Mineko Maeda (Osaka) Hiroshi Ochiai (Hokkaido) Tamao Saito (Hokkaido) Rob Kay (Cambridge) Jeff Williams(London) - from whom further information can be obtained E-MAIL jeff.williams@ucl.ac.uk Appendix: A More Detailed Project plan 1). The cDNA libraries The cDNA was synthesised using mRNA isolated from slugs, cloned into a small ampicillin resistance plasmid, transformed into E. coli and is now being gridded out in micro-titre plates. A total of 10-20,000 clones will be analysed. 2) Detection of high abundance sequences and avoidance of duplicating sequence determinations between the libraries. The cDNA clones will be replicated on to filters, at high density, which will then be hybridised with labelled slug cDNA. From the most strongly hybridising clones we will sequence only a limited number of randomly picked clones. In this way we will avoid massively redundant tag sequencing of the same, highly abundant mRNAs. While it will be impossible to entirely prevent such repetitive sequencing, it will actually be useful to sequence the same mRNA several times to ensure accuracy (typically the same region of a DNA is read 5 times in "shotgun" sequencing approaches, such as that proposed in the most recently suggested genomic sequencing plan from Bill Loomis). The total number of clones eventually sequenced will be between 10 and 20, 000 and this number should ensure a high (at least 50%) coverage of the mRNAs expressed at the slug stage. 3) Determination of the 5' and 3' sequences The cDNA inserts will be "tag" sequenced from their peripheries using primers located in vector sequences. This should give up to 1,000 nucleotides of sequence and, because the average mRNA in Dictyostelium is about 1.5Kb in length, over half of the Dictyostelium coding sequence present within the library will be established. These partial sequences will be searched against the databases and the cDNA clones will be prioritised for further analysis. Those that derive from known Dictyostelium genes will not be sequenced and those that encode "housekeeping" proteins (e.g. metabolic enzymes) will be given lowest priority. Those that encode entirely unknown proteins will be given intermediate priority and those that contain known sequence domains will have the highest priority. 4) Completion of the sequences We are committed to completing the entire sequence of each of the cDNAs. The Dictyostelium genome is closely mapped, therefore one of the major applications of EST's is rendered redundant. Also, in order to be useful as a gridded library, the entire sequence of each clone must be lodged in the database. Where necessary, we will therefore synthesise primers to complete the sequence of each cDNA. The commercial cost of primers has dropped dramatically in recent times and this is a viable proposition. =========== Abstracts =========== Identification of the Cell Fate Gene Stalky in Dictyostelium Wen-Tsan Chang, Peter C. Newell and Julian D. Gross Department of Biochemistry, University of Oxford, South Parks Road Oxford OX1 3QU, U.K. CELL, In Press SUMMARY Using insertional mutagenesis we have isolated a "stalky" mutant, in which cells normally destined to become spores end up as stalk cells. Similar mutants with this phenotype were previously observed after chemical mutagenesis but the gene could not be isolated. Our mutant, like the previous ones, is in stkA, Its defect, which is cell autonomous, is in spore formation and is not overcome by overexpressing cAMP-dependent protein kinase. The stkA gene is strongly expressed in the prespore region of aggregates, and not detectably expressed in the anterior prestalk (pstA) zone. The mutant expresses normal levels of early prespore-cell-specific transcripts but fails to produce the late prespore-specific transcript spiA. Rescue and sequencing of stkA reveals that it encodes a predicted 99-kD protein (STKA) with two putative C4 zinc fingers, one of which is a GATA-type zinc finger, indicating that it may be a transcription factor. This conclusion is supported by localisation of STKA in the nucleus. --------------------------------------------------------------------- Classification of Tyrosine Kinases from Dictyostelium discoideum with two distinct, complete or incomplete catalytic domains Kristin Adler, Gunther Gerisch, Ulrike von Hugo, Andrei Lupas, and Anton Schweiger FEBS Letters, in press Abstract: Two new kinases of Dictyostelium discoideum were identified by screening of a Lambda-gt11 expression library with a phosphotyrosine specific antibody. Amino-acid sequences derived from cDNA and genomic clones indicate that DPYK3 is a protein of 150 kDa and DPYK4 of 75 kDa. C-terminal fragments of each protein were produced in E. coli and shown to be autocatalytically phosphorylated at tyrosine residues. A common feature of these kinases is the presence of two different sequence stretches in tandem that are related to kinase catalytic domains. The sequence relationships of DPYK3 and 4 to other protein kinases, and the positions of their catalytic domain sequences within the phylogenetic tree of protein kinases were analysed. Domains I of both kinases and domain II of DPYK3 constitute, together with the catalytic domains of two previously described tyrosine kinases of D. discoideum, a branch of their own, separate from the tyrosine kinase domains in sensu strictu. Domain II in DPYK4 is found on a different branch close to serine/threonine kinases. --------------------------------------------------------------------- A NOVEL STRUCTURAL COMPONENT OF THE DICTYOSTELIUM CENTROSOME Astrid Kalt and Manfred Schliwa Adolf-Butenandt-Institutm Zellbiologie, University of Munich, Germany Summary The microtubule-organizing center of D. discoideum is an nucleus-associated body (NAB) that consists of a multilayered, box-shaped core embedded in an amorphous corona from which the microtubules emerge. The composition of the NAB is still largely unresolved. Here we have examined a high molecular weight component of the NAB which was identified by a monoclonal antibody raised against isolated nucleu/NAB complexes. This antibody recognized a 350 kDa component which is immunologically related to the D. discoideum heavy chain of myosin II. The 350-kDa antigen was localized only at the NAB in interphase cells, while in mitotic cells it was also found in the vicinity of the NAB as well as in association with the mitotic spindle. Immunogold labeling experiments showed that the protein is part of the NAB corona. This association was not destroyed by treatments with 2 M urea or 0.6 M KCL. The 350-kDa antigen was part of thiabendazole-induced cytoplasmic microtubule-organizing centers. A direct role in the polymerization of tubulin could not be determined in an in vitro-microtubule nucleation assay, whereas antibody electroporation of live cells appeared to interfere with the generation of a normal microtubule system in a subset of cells. Our observations suggest that the 350-kDa antigen is a structural component of the NAB corona which could be involved in its stabilization. --------------------------------------------------------------------- Adenylyl cyclase G, an osmosensor controling germination of Dictyostelium spores Saskia van Es1, Kiran J. Virdy2, Geoffrey S. Pitt3, Marcel Meima1, Todd W. Sands2, Peter N. Devreotes3, David A. Cotter2 and Pauline Schaap1. 1.Cell Biology Section, Institute for Molecular Plant Sciences, University of Leiden, Wassenaarseweg 64, 2333AL Leiden, The Netherlands. 2.Department of Biological Sciences, University of Windsor, Windsor, Ontario N9B3P4, Canada 3.Department of Biological Chemistry, Johns Hop kins University Medical School, Baltimore, Maryland 21205, USA. J. Biol. Chem. in press. SUMMARY Dictyostelium cells express a G-protein coupled adenylyl cyclase, ACA, during aggregation and an atypical adenylyl cyclase, ACG, in mature spores. The ACG gene was disrupted by homologous recombination. acg- cells developed into normal fruiting bodies with viable spores, but spore germination was no longer inhibited by high osmolarity, a fairly universal constraint for spore and seed germination. ACG activity, measured in aca-/ACG cells, was strongly stimulated by high osmolarity with optimal stimulation occurring at 200 milliosmolar. RdeC mutants, which display unrestrained protein kinase A (PKA) activity and a cell line, which overexpresses PKA under a prespore specific promoter, germinate very poorly, both at high and low osmolarity. These data indicate that ACG is an osmosensor controling spore germination through activation of protein kinase A. --------------------------------------------------------------------- Two ras genes in Dictyostelium minutum show high sequence homology, but different developmental regulation from Dictyostelium discoideum rasD and rasG genes Saskia van Es, Rolf A. Kooistra and Pauline Schaap Cell Biology Section, Institute for Molecular Plant Sciences, University of Leiden, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. Gene, in press. SUMMARY The social amoeba Dictyostelium discoideum expresses five ras genes at different stages of development. One of them, DdrasD is expressed during postaggregative development and tra nscription is induced by extracellular cAMP. A homologue of DdrasD, the DdrasG gene, is expressed exclusively during vegetative growth. We cloned two ras homologues, Dmras1 and Dmras2, from the primitive species D. minutum, which show high homology to DdrasD and DdrasG and less homology to the other Ddras genes. In contrast to the DdrasD and DdrasG genes, both the Dmras1 and Dmras2 genes are expressed during the entire course of development. The expression levels are low during growth, increase at t he onset of starvation and do not decrease until fruiting bodies have formed. Expression of neither Dmras1 nor Dmras2 is regulated by cAMP. So even though the high degree of homology between the ras genes of different species suggests conservation of function, this function is apparently not associated with a specific developmental stage. --------------------------------------------------------------------- WILDTYPE STRAINS OF DICTYOSTELIUM DISCOIDEUM CAN BE TRANSFORMED USING A NOVEL SELECTION CASSETTE DRIVEN BY THE PROMOTER OF THE RIBOSOMAL V18 GENE Birgit Wetterauer1, Piero Morandini2, Irene Hribar1, Irene Murgia-Morandini2, Ursula Hamker1, Charles Singleton3, and Harry K. MacWilliams1 ABSTRACT Almost all methods for transformation of the social ameba Dictyostelium discoideum rely on axenic growth, that is, growth in a synthetic medium, either throughout the procedure or for the transfection and a period of preselection. Axenic growth requires several mutations (Williams et al., 1974, Clarke et al., 1987). Here we describe a procedure which can be used to transform wildtype strains, which are able to grow only on the natural food source, bacteria. The method relies on a new selection cassette driven by the V18 promoter, a promoter which we show is substantially more active during growth on bacteria than the actin6 promoter, which is widely used for axenic transformation. The procedure gives transformation frequencies of about 10-5 with both strains Ax2 (capable of axenic growth) and NC4 (capable of growth only on bacteria). Using this vector, we have obtained NC4 strains carrying several beta-galactosidase reporter cassettes. Our vector can also be used in axenic transformations. --------------------------------------------------------------------- [End CSM-News, volume 7, number 9]