dictyNews Electronic Edition Volume 30, number 8 February 29, 2008 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ========= Abstracts ========= Establishment of a new method for precisely determining the functions of individual mitochondrial genes, using Dictyostelium cells Junji Chida 1,2, Aiko Amagai 1, Masashi Tanaka3 and Yasuo Maeda 1,* 1: Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan 2: Division of Enzyme Chemistry, Institute for Enzyme Research, Tokushima University, Tokushima, Tokushima 770-8503, Japan 3: Genomics for Longevity and Health, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan *Corresponding author BMC Genetics, in press Background: Disruption of mitochondrial genes may become a powerful tool for elucidating precisely the functions of individual mitochondrial genes. However, it is generally difficult to manipulate genetically mitochondrial genes, because 1) a mitochondrion is surrounded by inner and outer membranes, and 2) there are a large number of mtDNA copies in a single cell. This is the reason why we tried to establish a novel method for disrupting a certain mitochondrial gene (rps4), using Dictyostelium cells. Results: Here, we have developed a new method for specifically disrupting a mitochondrial gene (rps4 ; ribosomal protein subunit S4), by a combination of homologous recombination and delivery of an appropriate restriction endonuclease (SfoI) into mitochondria. First, mitochondrially targeted SfoI whose expression is under control of the tetracycline (Tet)-regulated gene expression system were introduced into cells heteroplasmic with respect to the rps4 gene. Then, the heteroplasmic cells were produced by homologous recombination by use of the construct in which the unique SfoI site and the 5'-half of the rps4 coding region was deleted not to be digested by SfoI, and therefore their mitochondria have both the wild-type mtDNA and the mutant mtDNA with the disrupted rps4 gene. In response to removal of Tet from growth medium, SfoI was selectively delivered into mitochondria and digested only the wild-type mtDNA but not the mutated rps4. Thus one can gain rps4-null cells with only the mutated mtDNA, under the Tet-minus condition. Conclusions: The mitochondrial gene-disruption method presented here must be widely useful for precisely determining the functions of individual mitochondrial genes. This is the first report to demonstrate complete and specific mitochondrial gene disruption. Submitted by: Yasuo Maeda [ymaeda@mail.tains.tohoku.ac.jp] -------------------------------------------------------------------------------- Dictyostelium Aurora kinase has properties of both Aurora A and Aurora B kinases. Hui Li, Qian Chen, Markus Kaller, Wolfgang Nellen, Ralph Graf & Arturo De Lozanne Eukaryotic Cell, accepted Aurora kinases are highly conserved proteins with important roles in mitosis. Metazoans contain two kinases, Aurora-A and B, that contribute distinct functions at the spindle poles and the equatorial region respectively. It is not currently known whether the specialized functions of the two kinases arose after their duplication in animal cells or were already present in their ancestral kinase. We show that Dictyostelium contains a single Aurora kinase, DdAurora, that displays characteristics of both Aurora-A and B. Like Aurora-A, DdAurora has an extended N-terminal domain with an A-box sequence and localizes at the spindle poles during early mitosis. Like Aurora-B, DdAurora binds to its partner DdINCENP and localizes on centromeres at metaphase, the central spindle during anaphase and the cleavage furrow at the end of cytokinesis. DdAurora also has several unusual properties. DdAurora remains associated with centromeres in anaphase and this association does not require an interaction with DdINCENP. DdAurora then localizes at the cleavage furrow but only at the end of cytokinesis. This localization is dependent on DdINCENP and the motor proteins Kif12 and myosin II. Thus, DdAurora may represent the ancestral kinase that gave rise to the different Aurora kinases in animals and also those in other organisms. Submitted by: Arturo De Lozanne [a.delozanne@mail.utexas.edu] -------------------------------------------------------------------------------- An anatomy ontology to represent biological knowledge in Dictyostelium discoideum Pascale Gaudet1*, Jeffery G. Williams2, Petra Fey1, Rex L. Chisholm1 1dictyBase, Northwestern University, Center for Genetic Medicine, 676 N. St.Clair, Chicago, IL, 60611, USA. 2School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom. *Corresponding author BMC Genomics, accepted Dictyostelium discoideum is a model system for studying many important physiological processes including chemotaxis, phagocytosis, and signal transduction. The recent sequencing of the genome has revealed the presence of over 12,500 protein-coding genes. The model organism database dictyBase hosts the genome sequence as well as a large amount of manually curated information. We present here an anatomy ontology for Dictyostelium based upon the life cycle of the organism. Anatomy ontologies are necessary to annotate species-specific events such as phenotypes, and the Dictyostelium anatomy ontology provides an essential tool for curation of the Dictyostelium genome. Submitted by: Pascale Gaudet [pgaudet@northwestern.edu] ============================================================== [End dictyNews, volume 30, number 8]