Dicty News Electronic Edition Volume 15, number 10 November 18, 2000 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.northwestern.edu/dicty" Editors note: The following is a draft proposal developed by a committee constituted at the Dundee Dicty 2000 meeting to develop a proposal for genetic nomenclature. The committee is anxious for feedback. To facilitate the widest possible involvement of the community in this important discussion, please send your comments to the email server address: dicty@listserv.it.northwestern.edu. ============================================== Proposal for standard genetic nomenclature ============================================== Dictyostelium Genetic Nomenclature and Strain designations Draft. Nov 15, 2000. Introduction The purpose of the proposed nomenclature system is to give a compact, accurate description of Dictyostelium strains and loci. We present this proposal to stimulate the universal adoption of a uniform nomenclature that we believe will facilitate everyone’s work in the long term. Have you ever received a strain marked ‘geneX-KO’, perhaps through a third party, and not been able to tell what the parent is, what the selectable marker is, what manner of KO it has, or which laboratory made it? This situation can get a lot worse, as strains with multiple KO’s are made in a variety of backgrounds, as interaction genetics by library complementation leads to the re-appearance of chemical mutants, and as gene replacement gives us a variety of temperature-sensitive and dominant alleles of known genes. Several laboratories have noticed effects of genetic background on mutant phenotype made in Ax2, Ax3, DH1, and so on. As more quantitative measures of phenotype are used this is going to become an increasing problem. Finally, we hope that a Strain Repository will be established to preserve and distribute mutant strains. This will only be feasible if the strains in it can be accurately described and traced. For these reasons, we feel that it is essential to have a uniform nomenclature system that everybody can use and which will help in the organization and distribution of strains in a repository. There are two conflicting pressures on gene nomenclature: the need to be compact and unambiguous, mainly for purposes within the field, and the need to be accessible across fields. The Demerec nomenclature, which is already in use in the field, is compact and accurate. We recommend that Demerec nomenclature be adopted, but with suitable modifications. Many Demerec gene abbreviations are not necessarily recognizable to outside workers and they may not connect Dictyostelium work to other work on similar, conserved genes. One way of bridging this gap is to use the ‘standard’ name for a protein and gene where necessary in descriptive sections of papers or grants. Thus we have the GSK3, STATa, PI3K proteins, and can talk about their genes as the GSK3, STATa genes, if we wish. But these genes will also have Demerec names gskA, dstA etc. By putting the ‘standard’ name in the Title, Keywords, Abstract, Text and Figure legends of a paper, we can ensure that it is accessible to computer searching as well as to readers outside the field. The formal gene names will be in the Materials and Methods, strain genotypes and various gene index databases that will provide the "Rosetta Stone" for all names. Although this suggestion leads to redundancy and some possibility of confusion, we feel that it reflects the reality of the gradual merger of gene naming systems drawn from different organisms and from biochemistry, which is the inevitable result of the conservation of many genes among organisms. It also reflects the reality of usage by many Dictyostelium laboratories. Strain naming is done for purposes within the field and so does not suffer from the same conflicts as gene naming. However we feel that it would be useful if it could be done in a standard way which indicates the laboratory of origin. This is the standard practice in most bacterial and yeast genetics laboratories. In our field, the practice for many laboratories is to use a 2- or 3-letter code unique to each laboratory, which is used to designate all of their strains. A list of these codes will be available on the web and laboratories can ‘register’ new ones through the proposed nomenclature committee. We recommend that a strain table be included in papers that employ multiple strains. Thus, some of the more cumbersome names proposed below should be included once in the table and a shorter form of the named used in the paper (with reference to the Table the first time the abbreviated name is used). In a few years, if we can develop a strain repository, it will be possible to have a column in such a table that has a repository number for each of the most important strains in a paper. This device will improve the ability of workers in the field and outside the field to locate original strains, obtain them from a strain repository, and to repeat experiments with rigor. We expect Dictyostelium to have about 10,000 genes, and the sequence for most of these should be available soon through the sequencing projects. Once these have been annotated they will be given temporary names (‘homologous to yeast gene YC1234’), which will be merged with the Demerec names, as more information becomes available. Formal rules and requirements for naming or re-naming genes will be discussed by all at a future date, but we suggest that individuals name genes only after they have generated significant new information about a gene within the context of a formal publication. Two obvious ways to do this would be to describe a mutation a given gene and report the results in a publication, or to compare a gene (or gene family) with genes in other species and publish those studies in a review article or as part of a primary research paper. The detailed recommendations of the nomenclature committee set up at the Dundee International Meeting are given below. We also propose setting up a clearing house for registering gene names and laboratory strain codes. For now, all laboratories with strain prefixes should inform Adam Kuspa so that the nomenclature committee can collate the information in the interim before a clearing house is established. Avoiding duplicate gene names may be difficult before the clearing house is set up, but curators of the large gene databases in Tsukuba, San Diego, Hinxton, Houston and Cologne/Jena should be able to offer some guidance. We welcome ideas and suggestions on this draft proposal, including missed references and suggestions for questions (and answers) under the FAQ section that will be attached to the final document. Much of the committee’s discussion focused on how to name genes. Views on this point differed, so in composing the draft below the committee had to weigh the need to present a concrete proposal, with the desire to present possible variations and additions. We made the decision to present the most compact gene nomenclature in the draft, but added an addendum at the end that expresses additions to the basic proposal which the committee also considered. The body of the proposal was endorsed by the majority of the committee, while the addendum was supported by a minority. We thank Jacob Franke for suggestions and careful review of this draft. Adam Kuspa (Chairman) Ludwig Eichinger Robert R. Kay Alan R. Kimmel Hideko Urushihara Richard Kessin (ex officio) Chairman, Dictyostelium Community Organizing Committee. Genes Gene names should be devised to conform to the following conventions derived from Demerec et al. (1966). A locus descriptor consisting of 3 italic letters, followed by a capital italicised letter to distinguish genes with the same descriptor that are related in a significant way. Thus, related genes should have the same 3-letter descriptor and successive capital letters (rdeA, rdeB and rdeC; or tagA, tagB and tagC). Gene names that do not follow these rules do exist (e.g. act6, act15, pyr5-6 and erk1), but the original naming authors can change any gene name, if they wish, by describing the name change in their next publication containing the gene and informing the clearinghouse. For example, "The spore coat protein gene cotB (formerly referred to as 'SP70') was introduced under the control of..." This change was made by Bill Loomis in 1990 (but not in these exact words). In the absence of formal renaming, the system will retain all extant names. Mutant alleles. All necessary details about an allele should be published somewhere (for example in a strain table), but need not be carried in its name. Since there are a large number of possible alleles of a gene, authors should use any unique allele name. Since a one-, two-, three- or four-digit number cannot be expected to remain unique without considerable effort by a clearinghouse, more detailed names should be used. To indicate an allele, as opposed to another gene, allele names should be placed in parentheses and in italics following the gene name without a space, as in yakA(AK235) and yakA(AK800), which are two different insertion mutations in yakA named for the strain in which they were isolated. Where the nature of the mutation is not known or only a single allele is relevant within a given document (paper or grant), a superscript minus sign should be used for brevity (regA-). The use of superscripts to describe the general nature of an allele can be used, as in regA(AK202ts) or regAts, but should be limited to two or three letters, e.g. ts, cs (cold sensitive), hc (high copy) or dn (dominant negative). Amino acid substitutions should be given as the old residue in single-letter code with its codon location followed by the new (mutant) amino acid, as in regA(D212A) which has an alanine in place of the aspartate at position 212. The amino acid change is an interpretation of what the gene will produce and refers to a protein so it is not put in italics. Ax4 is the reference strain for the "wild-type" amino acid sequence within proteins since it will be the first strain sequenced. Proteins A protein should be named after the gene encoding it by capitalizing the first letter and without the use of italics. Thus, the protein encoded by regA is RegA, or RegA(D212A) in the case of a mutant protein. When naming a protein before its gene is cloned, one should keep the genetic nomenclature in mind. For example, the name PSF for Clarke’s Pre-starvation Factor can easily be changed to "PsfA" when the psfA gene is identified, but "p38" would be more problematic and likely necessitate renaming the protein when the primary sequence is known. Phenotypes No convention for describing phenotypes of Dictyostelium genes has been invoked, but when used they should not be italicized. Three letter abbreviations of phenotypes can be used to name genes once the relevant genes are cloned, characterized and found to have a novel structure (e.g. tagA for the "tip-less aggregate" phenotype). The following names should be used to indicate common drug resistance/sensitivity and nutrient auxotrophy/prototrophy. For clarity, relevant phenotypic markers should be included at the end of formal genotypes (no italics): neor (indicates a neomycin phosphotransferase gene is functional). neos (for clarity, or when G418 selection on a neor strain is relaxed and G418-sensitive strain is isolated). thy- (indicates thy1 is mutant and the strain requires thymidine). thy+ (used when a thy1 mutant has been complemented). bsr (indicates the blasticidin resistance gene bsr is present). bss (for clarity, or when bsr has been introduced but is non-functional). ura+ (uracil independent. Note that this can be different from pyr5-6+). ura- (most often used to indicate pyr5-6-). foar (most often this will be redundant with ura-). hygr (hygromycin resistance). bler (bleomycin resistance). Genotypes Genotypes should describe the relevant genetic modifications present in a given strain. Genes listed in a genotype are considered to by mutant in some way. Every genetic element described in a genotype (gene, plasmid, DNA fragment) should be separated by a comma. To distinguish chromosomal loci from exogenous genes, every gene or construct contained on DNA that was introduced into cells by transformation should be listed within brackets regardless of whether that DNA is carried on a plasmid, integrated as a fragment or was amplified by replication once inside cells. Molecular genetic constructs Reporter genes and gene fusions should be named with the relevant components separated by slashes and dashes to indicate DNA fusion, as follows. Typically, the components will be a promoter, a coding sequence or ORF encoding a reporter protein. Promoters (± a few codons) should be separated from coding sequence by a slash (/) and coding sequences separated by dashes (-). So a Talin GFP fusion under the transcriptional control of a cotB promoter would be written: cotB/talA-gfp. When a coding region is under the transcriptional control of the native promoter it could be written talA/talA-gfp to emphasize this, or simply written as talA-gfp. Of course, the more commonly used GFP variant would be written talA-gfp(S65T). Constructs carried within cells should be described in the formal genotype of that strain and placed in brackets. Thus, a cotB promoter b-galactosidase reporter construct in a mutant strain might have a formal genotype of "regA(AK202ts), p88d1[cotB/lacZ] neor", but could be shortened to "regA-[cotB/lacZ]" in the context of a sentence describing the strain’s properties in the results section of a paper. Again, strain tables add clarity. Plasmids Naturally occurring plasmids are named by a prefix indicating the genus and species, as in Ddp1. Derivatives of the natural plasmids and other shuttle vectors should be indicated with a lowercase "p" prefix (pDXA-3C). For genes on a plasmid, and any other gene that is introduced into a strain by experiment (see below), use the same naming system as for chromosomal genes, but placed within square brackets (e.g. pDneo67[act6/regA] to indicate a regA coding sequence fused to the promoter of the actin 6 gene on plasmid pDneo67). Strains Strains available for distribution must have an unambiguous name, consisting of 2 or 3 capitals plus a unique serial number (e.g. HM1 or HTY217). Labs or workers should always use the same capital prefixes or small group of prefixes. The prefixes are assigned by a clearinghouse upon request. See Appendix 1 for a current list of assigned strain prefixes. In the past all lab designations started with either H (for haploid) or D (for diploid) and many of these strains are still in use. Management. A clearinghouse will be established to ensure that lab codes and gene names are not duplicated. Until the 2001 international meeting in San Diego, the clearinghouse will be the nomenclature committee, headed by Adam Kuspa. Contact the committee through him via E-mail at akuspa@bcm.tmc.edu. The membership of a standing nomenclature committee can be determined at the 2001 meeting. Frequently Asked Questions When should genes be named? In general, genes should be named after they are completely characterized structurally and after some functional information is obtained by mutation, expression, or by purification of a gene product. Genes could also be named by virtue of their being nearly identical to another Dictyostelium gene product or a gene described in another species. For example, 50% amino acid identity from N to C terminus between a new Dictyostelium protein and a protein described in another species may be considered sufficient grounds for using the previously described gene name (suitably modified) for the Dictyostelium gene. As a practical matter, the likely key decision point for naming a gene will come when it is time to submit a gene to a database (GenBank/DDBJ/EMBL). Authors should be judicious about giving the sequence a name that they expect to use in publications since these databases are the way that many researchers first come in contact with a gene from another species. Nothing will be more confusing than to have genes named one way in GenBank and another way in the literature. Will the nomenclature committee re-name genes that do not conform to Demerec? No. The original naming authors can formally re-name genes if they wish, as described above. Future interspecies genome comparisons may require re-naming some genes, but the manner in which such issues will be dealt with is not presently clear. References General: Demerec, M. et al., (1966). A proposal for a uniform nomenclature in bacterial genetics. Genetics 54:61-76. Kay, R.R., W.F. Loomis and P.N. Devreotes (1998). Genetic Nomenclature Guide. Elsevier Science Ltd. London. pp S.5-S.6 (TIGS supplement) Gene Lists: DictyDB http://glamdring.ucsd.edu/others/dsmith/dictydb.html#C Kuspa, A., D. Maghakian, P. Bergesch, and W.F. Loomis. 1992. Physical mapping of genes to specific chromosomes in Dictyostelium discoideum. Genomics 13:49-61. Loomis, W.F., D. Welker, J. Hughes, D. Maghakian, and A. Kuspa. 1995. Integrated maps of the chromosomes in Dictyostelium discoideum. Genetics 141:147-157. Kuspa, A., and W.F. Loomis. 1996. Ordered yeast artificial chromosome clones representing the Dictyostelium discoideum genome. Proc. Natl. Acad. Sci. USA 93:5562-5566. Appendix 1. List of Known Strain Prefixes by Laboratory DP, NP and XP; P. Newell HM and DM; R.R. Kay HL, DL and DG; W.F. Loomis GS; Gad Shaulsky AK, V and W; A. Kuspa KY; K. Yanagisawa HU, DUK; K. Williams HUD, DUD; D. Welker HC, DCB; B. Coukell HPF, DPFP; P. Fisher HH, DH, HR (Kessin, Ennis) Addendum: While we recognize that Demerec nomenclature is compact, accurate and uniform, we also realize that it is not necessarily accessible to other areas or systems. One problem with Demerec is that it is fixed at 3 letters and does not permit numbers or Greek letters. So STAT, GSK3, PI3K, and Gb are not accommodated by Demerec. Many Demerec abbreviations are already used in Dictyostelium for genes that are conserved in other systems. Unfortunately, they may not be recognizable to outside workers or fail to be connected to similar, conserved genes in database searches. In such cases, the best way to bridge this gap is to use the standard universal (non-Demerec) name for a protein and gene where appropriate. Genes can thus be written directly as GSK3, STATa, PI3K, Gb (all in italics). These genes will also have Demerec names but, in such cases, the Demerec names will be relegated to Materials and Methods and Dictyostelium websites. In websites, the genes will be cross referenced to their universal (non-Demerec) name. For non-Demerec usage within manuscripts, etc. there can be a modification for clarity, based upon mammalian usage. Wild-type genes are denoted in capital italicized letters, (eg. PI3K), with mutants in lower case (eg pi3k). We recognize one major difficulty. There will be genes with Demerec names, but non-Demerec orthologs. The same gene will be called different names by different authors. For uniformity, the standard name should take preference. Duality can be acknowledged parenthetically in an introduction. Situations already exist in Dictyostelium and other systems where identical genes have more than one name. ========================= Postdoctoral Position ========================= POSTDOCTORAL POSITIONS IN FUNCTIONAL GENOMICS Postdoctoral positions are available to work on the Dictyostelium functional genomics program at Baylor College of Medicine. Individuals interested in large-scale mutant characterization, expression arrays, genome sequence analysis or the development of data mining and visualization tools are invited to apply. Baylor College of Medicine is part of the Texas Medical Center in Houston, one of the largest and most rapidly developing medical centers in the world. Baylor offers competitive salaries and benefits as well as great employment opportunities for spouses. We expect to fill several positions in 2001. Candidates holding a Ph.D. degree, or equivalent, in relevant fields should submit a short description of research interests, cv and a list of 3 references to the appropriate faculty member listed below. Adam Kuspa (Genome sequencing, Gene function) akuspa@bcm.tmc.edu Gad Shaulsky (Expression arrays, Gene function) gadi@bcm.tmc.edu John Halter (Computational modeling, data mining) jah@bcm.tmc.edu Jeff Tollett (Large-scale phenotyping and expression profiling) jtollett@bcm.tmc.edu Chad Shaw (Statistical analyses and modeling) cashaw@bcm.tmc.edu ============== Abstracts ============== Adherens junctions and beta-catenin mediated cell signalling in a non-metazoan organism Mark J. Grimson, Juliet C. Coates , Jonathan P. Reynolds, Mark Shipman Richard L. Blanton & Adrian J. Harwood Nature (in press) Abstract Mechanical forces between cells play a major part in the organisation of animal tissues. Adherens junctions are an important component of these tissues, connecting cells via their actin cytoskeleton and allowing assembly of tensile structures. At least one adherens junction protein, beta-catenin, also acts as a signalling molecule, directly regulating gene expression. To date, adherens junctions have only been detected in metazoa, so to examine their evolutionary origins, we sought them outside the animal kingdom. The non-metazoan Dictyostelium discoideum forms a multicellular, differentiated structure. Here, we describe the discovery of actin-associated intercellular junctions in Dictyostelium. Furthermore, we have isolated a gene encoding a beta-catenin homologue, aardvark, which is a component of the junctional complex and, independently, is required for cell signalling. This is the first report of adherens junctions outside the animal kingdom and demonstrates that the dual role of beta-catenin in cell-cell adhesion and cell signalling evolved prior to the origins of metazoa. ---------------------------------------------------------------------------- A genetic interaction between a ubiquitin-like protein and ubiquitin-mediated proteolysis in Dictyostelium discoideum. Stefan Pukatzki*, Herbert L. Ennis, and Richard H. Kessin Department of Anatomy and Cell Biology, College of Physicians & Surgeons, Columbia University, New York, NY 10032. * Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115. Biochimica et Biophysica Acta, in press Abstract A ubiquitination factor, NosA, is essential for cellular differentiation in Dictyostelium discoideum. In the absence of nosA, development is blocked, resulting in a developmental arrest at the tight-aggregate stage, when cells differentiate into two precursor cell types, prespore and prestalk cells. Development is restored when a second gene, encoding the ubiquitin-like protein SonA, is inactivated in nosA-mutant cells. SonA has homology over its entire length to Dsk2 from Saccharomyces cerevisiae, a ubiquitin-like protein that is involved in the assembly of the spindle pole body. Dsk2 and SonA are both stable proteins that do not seem to be subjected to degradation via the ubiquitin pathway. SonA does not become ubiquitinated and the intracellular levels of SonA are not affected by the absence of NosA. The high degree of suppression suggests that SonA rescues most or all of the defects caused by the absence of nosA. We propose that NosA and SonA act in concert to control the activity of a developmental regulator that must be deactivated for cells to cross a developmental boundary. ---------------------------------------------------------------------------- Percolation clustering: a novel approach to the clustering of gene expression patterns in Dictyostelium development. Roman Sasik, Terry Hwa, Negin Iranfar, and William F. Loomis Department of Physics and Division of Biology University of California at San Diego, La Jolla, CA 92093, USA Pacific Symp. Biocomputing (in press) Abstract We present a novel approach to the clustering of gene expression patterns based on the mutual connectivity of the patterns. Unlike certain widely used methods (e.g., self-organizing maps and K-means) which essentially force gene expression data into a fixed number of predetermined clustering structures, our approach aims to reveal the natural tendency of the data to cluster, in analogy to the physical phenomenon of percolation. The approach is probabilistic in nature, and as such accommodates the possibility that one gene participates in multiple clusters. The result is cast in terms of the connectivity of each gene to a certain number of (significant) clusters. A computationally efficient algorithm is developed to implement our approach. Performance of the method is illustrated by clustering both constructed data and gene expression data obtained from Dictyostelium development. ---------------------------------------------------------------------------- INDUCIBLE EXPRESSION OF EXOGENOUS GENES IN DICTYOSTELIUM DISCOIDEUM USING THE RIBONUCLEOTIDE REDUCTASE PROMOTER Pascale Gaudet1,2, Harry MacWilliams4 and Adrian Tsang1,2,3* 1Department of Chemistry and Biochemistry, 2Centre for Structural and Functional Genomics, 3Department of Biology, Concordia University, 1455 de Maisonneuve Blvd. W., Montreal, Quebec, H3G 1M8, Canada 4Zoologisches Institut, Ludwig-Maximilians-Universität, Luisenstrasse 14, 80333 München, Germany Nucleic Acids Research, in press ABSTRACT We report here the development of a regulated gene expression system for Dictyostelium discoideum based on the DNA-damage inducibility of the rnrB gene. rnrB, which codes for the small subunit of the enzyme ribonucleotide reductase, responds to DNA-damaging agents at all stages of the D. discoideum life cycle. Doses that have little effect on development have previously been shown to increase the level of the rnrB transcript by up to 15-fold. Here we show that all elements necessary for DNA-damage induction are contained in a 450-bp promoter fragment. We used a fusion of the rnrB promoter with the gene encoding GFP to demonstrate an up to 10-fold induction at the RNA level, which appears in all aspects similar to the induction of the endogenous rnrB transcript. Using a fusion with the lacZ gene we observed an up to 7-fold induction at the protein level. These results indicate that the rnrB promoter can be used to regulate the expression of specific genes in D. discoideum. This controllable gene expression system provides the following new characteristics: the induction is rapid, taking place in the order of minutes and the promoter is responsive at all stages of the D. discoideum life cycle. ---------------------------------------------------------------------------- [End Dicty News, volume 15, number 10]