Dicty News Electronic Edition Volume 12, number 10 May 8, 1999 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" ================ Announcement ================ Due in part to Robert Insall's move to Birmingham, the European mirror of the Dicty Web site no longer functions. It appears that the internet links between europe and the US are functioning well, so it is not clear we need the european mirror. We propose to dispense with the mirror. If you experience problems with you connections to the Dicty site, or have other thoughts regarding this please let us know at dicty@nwu.edu. ============== Abstracts ============== A myb-related protein required for culmination in Dictyostelium. Kunde Guo , Christophe Anjard , Adrian Harwood , Hyun-Ji Kim , Peter C. Newell and Julian D. Gross. Dept of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU. U.K. Development (in press). Abstract: The avian retroviral v-myb gene and its cellular homologues throughout the animal and plant kingdoms contain a conserved DNA binding domain. We have isolated an insertional mutant of Dictyostelium unable to switch from slug migration to fruiting body formation i.e. unable to culminate. The gene that is disrupted, mybC, codes for a protein with a myb-like domain that is recognized by an antibody against the v-myb repeat domain. During development of myb+ cells, mybC is expressed only in prestalk cells. When developed together with wild type cells mybC- cells are able to form both spores and stalk cells very efficiently. Their developmental defect is also bypassed by overexpressing cAMP-dependent protein kinase. However even when their defect is bypassed, mybC null slugs and culminates produce little if any of the intercellular signalling peptides SDF-1 and SDF-2 that are believed to be released by prestalk cells at culmination. We propose that the mybC gene product is required for an intercellular signaling process controlling maturation of stalk cells and spores and that SDF-1 and/or SDF-2 may be implicated in this process. ---------------------------------------------------------------------------- IDENTIFICATION OF CIS-REGULATING ELEMENTS AND TRANS-ACTING FACTORS REGULATING THE EXPRESSION OF THE GENE ENCODING THE SMALL SUBUNIT OF RIBONUCLEOTIDE REDUCTASE IN DICTYOSTELIUM DISCOIDEUM Claire Bonfils1,4, Pascale Gaudet2,3, and Adrian Tsang1,2,3 1Department of Biology, 2Department of Chemistry and Biochemistry, and 3Centre for Structural and Functional Genomics, Concordia University, Montreal, Quebec, H3G 1M8, Canada 4Present address: MethylGene Inc., 7220 Frederick-Banting, Ville St-Laurent, Quebec, H4S 2A1, Canada Journal of Biological Chemistry, accepted SUMMARY We have examined the promoter of rnrB, the gene encoding the small subunit of ribonucleotide reductase of Dictyostelium discoideum, using lacZ as a reporter gene. Deletion analysis showed that expression of this gene in vegetative cells involves an A/T-rich element whereas its expression in prespore cells during development requires a region encompassing two G/C-rich elements, designated box A and box B. Removal of boxes A and B results in very low level of activity. When either box A or box B is deleted, prestalk cells adjacent to the prespore zone also express _-galactosidase. The behaviour of these cis-regulatory elements implies that the mechanism regulating the prespore-specific expression of rnrB is different from that regulating other known prespore genes. We have used electrophoretic mobility shift assays to identify factors that interact with box A and box B. Box A interacts with a factor that is found in the nuclear fraction. While box B interacts with a factor that is present in the cytosolic fraction throughout growth and development, its presence in the nuclear fraction is developmentally regulated. Results from competition assays suggest that both box A and box B interact with transcriptional activators that have not been characterized previously. ---------------------------------------------------------------------------- Endocytic transit in Dictyostelium Markus Maniak MRC Laboratory for Molecular Cell Biology, University College London, London, UK Protoplasma, in press Summary. The cells of Dictyostelium are soil amoebae with a simple endocytic pathway: Particles or fluid are taken up at the plasma membrane in a process dependent on the actin cytoskeleton. After rapid acidification and subsequent neutralisation of the food vacuoles during which breakdown of the contents occurs, indigestible remnants are exocytosed. This tight coupling between endocytosis and exocytosis is thought to maintain membrane homeostasis. In spite of the apparent overall difference between the endocytic pathways of mammalian cells and Dictyostelium, conserved proteins are involved in individual steps of endocytic transport, possibly indicating that in mammalian cells it is only the routing of marker that has evolved from a simple transit to a complex, branched pathway. ---------------------------------------------------------------------------- Disturbed communication between actin- and nucleotide binding sites in a myosin II with truncated 50K/20K junction Menno L.W. Knetsch 1, Taro Q.P. Uyeda 2, and Dietmar J. Manstein 1 1)Max-Planck-Institute for Medical Research, Dept. of Biophysics, Jahnstrasse 29, D-69120 Heidelberg, Germany and 2) Bionic Design Group, National Institute for advanced Interdisciplinary Research, AIST, 1-1-4 Higashi, Tsukuba, Ibaraki 305, Japan J. Biol. Chem (in press.) ABSTRACT The kinetic and functional consequences of deleting nine residues from an actin-binding surface loop (loop 2) were examined to investigate the role of this region in myosin function. The nucleotide-binding properties of myosin were not altered by the deletion. However, the deletion affected actin binding and the communication between the actin- and nucleotide-binding sites. The affinity of M765NL for actin (644 nM) was approximately 100-fold lower than that of wild-type construct M765 (5.8 nM). Despite this reduction in affinity, actin binding weakened the affinity of ADP for the motor to a similar extent for both mutant and wild-type constructs. The addition of 0.5 mM actin decreased ADP affinity from 0.6 mM to 34 mM for M765NL and from 1.6 mM to 39 mM for M765. In contrast, communication between the actin- and nucleotide-binding sites appears disturbed in regard to Pi-release: thus, basal ATPase activity for M765NL (0.19 s-1) was 3-fold larger than for M765 (0.06 s-1) and the stimulation of ATPase activity by actin was 5-fold lower for M765NL. These results indicate different paths of communication between the actin- and nucleotide-binding sites, in regard to ADP- and Pi-release, and they confirm that loop 2 is involved in high affinity actin binding. ---------------------------------------------------------------------------- Ribosomal protein gene expression is cell type specific during development in Dictyostelium discoideum. Ameeta K. Agarwal, Susan N. Parrish and Daphne D. Blumberg Department of Biological Science, University of Maryland Baltimore County 1000 Hilltop Circle, Baltimore, Md. 21250 In press in Differentiation. ABSTRACT: Starvation for amino acids initiates the developmental cycle in the Cellular Slime Mold, Dictyostelium discoideum. Upon starvation one of the earliest developmental events is the selective loss of the ribosomal protein mRNAs from polysomes. This loss depends upon sequences in the 5’ non-translated leader of the r-protein mRNAs. Here evidence is presented which indicates that those cells which will become prestalk cells express the ribosomal protein genes during development under starvation conditions. Cells which enter the prespore pathway shut off r-protein synthesis. The promoter and 5’ non-translated leader sequences from two ribosomal protein genes, the rp-L11 and the rp-S9 genes, are fused to the E.coli -galactosidase reporter gene. While -galactosidase enzyme activity is detected in situ in most growing cells, by 15 hours of development -galactosidase enzyme activity is largely lost from the prespore cells although strong -galactosidase enzyme activity is present in the prestalk cells. These observations suggest the possibility that the ribosomal protein mRNAs are excluded from polysomes in a cell type specific manner. ---------------------------------------------------------------------------- Dictyostelium ribosomal protein genes and the elongation factor 1B gene show co-ordinate developmental regulation which is under post-transcriptional control. Ameeta K. Agarwal and Daphne D. Blumberg Department of Biological Science, University of Maryland Baltimore County 1000 Hilltop Circle, Baltimore, Md. 21250 In press in Differentiation ABSTRACT: Starvation for amino acids initiates the developmental program in the Cellular Slime Mold, Dictyostelium discoideum. One of the earliest developmental events is the decline in ribosomal protein synthesis. The ribosomal protein mRNAs are excluded from polysomes within 20 minutes to 1 hour following the removal of nutrients and their mRNA levels decline sharply at about 9 hours into the 24 hour developmental cycle. It has been generally assumed that the decline in r- protein mRNA levels during late development reflected a decline in the transcription rate. Here we demonstrate that this is not the case. The transcription rates of three ribosomal protein genes, rpL11, rpL23 and rpS9 as well as an elongation factor 1B gene have been determined during growth and development in Dictyostelium. Throughout growth and development the transcription rate of the ribosomal protein genes remains relatively constant at 0.2 to 0.5% of the rate of rRNA transcription while the elongation factor 1B gene is transcribed at 0.4 to 0.6% of the rRNA rate. This low but constant transcription rate is in contrast to a spore coat protein gene Psp D which is transcribed at 6% of the rRNA rate in late developing cells. The elongation factor 1B gene appears to be co-regulated with the ribosomal protein genes both in terms of its transcription rate and mRNA accumulation. Dictyostelium has been a popular model for understanding signal transduction and the growth to differentiation transition, thus it is of significance that the regulation of ribosome biosynthesis in Dictyostelium resembles that of higher eukaryotes in being regulated largely at the post-transcriptional level in response to starvation as opposed to yeasts where the regulation is largely transcriptional. ---------------------------------------------------------------------------- [End Dicty News, volume 12, number 10]