Total tetra knockout of GP138 multigene family implicated in cell interactions in Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum reproduces sexually under submerged and dark conditions. A cell surface glycoprotein gp138 has been identified as a target molecule for cell fusion-blocking antibodies, and is considered to be indispensable for the sexual cell fusion in this organism. Currently, four isoforms of gp138, DdFRP1alpha, DdFRP1beta, DdFRP2, and DdFRP3, are known. Genes encoding the latter three isoforms, GP138C, GP138A, and GP138B, have been isolated, comprising a GP138 multigene family. Here we isolated the fourth GP138 gene, GP138D, encoding DdFRP1alpha. These GP138 genes were found to cluster in a tandem array on chromosome 5, being bordered by two GP138-like sequences highly homologous to them but truncated. To clarify functional relationships among the GP138 family members, the entire GP138 region was deleted by a single knockout. Northern hybridization and western immuno-blotting analyses confirmed complete losses of GP138 mRNA and DdFRPs in the knockout strains, indicating that there are no more GP138 genes. Unexpectedly, however, the GP138-null mutants were fully potent for both sexual cell fusion and subsequent development. In addition, the original fusion-blocking antibodies detected a cell surface protein of close electrophoretic mobility to gp138 in the knockouts, suggesting the possibility that the actual target molecule of the fusion-blocking antibodies was not DdFRPs but this unidentified component. Since GP138-null mutants exhibited no obvious defects either in growth or asexual development, the real function of the GP138 family is unknown. Nevertheless, the expression levels of other developmental genes such as acaA, csaA, cotA-C, and spiA appeared to be altered in the GP138-null mutants. Therefore, it seems to have a non-critical but some role(s) during asexual development.     

Gene-specific expression of the actin multigene family of Dictyostelium discoideum

We have investigated the expression of 14 cloned genes of the 20-member actin multigene family of Dictyostelium discoideum using gene-specific mRNA complementary probes and an RNase protection assay. Actin gene expression was studied in vegetative cells and in cells at a number of developmental stages chosen to represent the known major shifts in actin mRNA and protein synthesis. At least 13 of these genes are expressed. A few genes are expressed very abundantly at 10% or more of total actin mRNA; however, the majority are maximally expressed at 1 to 5% of actin message. Although all of the genes are transcribed in vegetative cells, most genes appear to be independently regulated. Actin 8 appears to be transcribed at constant, high levels throughout growth and development. Actin 12 mRNA is maximally expressed in vegetative cells but the level is reduced appreciably by the earliest stage of development examined, while Actin 7 mRNA is specifically induced approximately sevenfold at this time. The rest of the genes appear to be induced 1.5 to 2-fold early in development, coincident with the increase in total actin mRNA. Since 12 of the genes code for extremely homologous proteins, it is possible that the large number of actin genes in Dictyostelium is utilized for precise regulation of the amount of actin produced at any stage of development, even though individual gene expression appears in some cases to be very stage-specific. In addition to these 13 actin genes, at least two and possibly four more genes are known to be expressed, because they are represented by complementary DNA clones, and an additional one or two expressed genes are indicated by primer extension experiments. Only one known gene, Actin 2-sub 2, is almost certainly a pseudogene. Thus the vast majority of Dictyostelium actin genes are expressed.     

A protein with homology to the C-terminal repeat sequence of Octopus rhodopsin and synaptophysin is a member of a multigene family in Dictyostelium discoideum

Monoclonal antibodies were raised against a protein with a molecular mass of 24 kDa that has been described as a membrane-associated, actin binding protein from Dictyostelium discoideum [( 1985) J. Cell Biol. 100, 727-735]. Using these monoclonal antibodies we isolated from a lambda gt11 expression library cDNA clones coding for this protein. The cDNA deduced amino acid sequence revealed the presence of an unusual carboxy-terminus which has homologies to the C-termini of Octopus rhodopsin and synaptophysin. This part of the protein sequence contains 5 direct repeats with the motif GYP (P)Q(P). Southern and Northern blots showed that this sequence is present in a series of Dictyostelium genes transcribed in all stages of development.     

The multigene immunophilin family of Dictyostelium discoideum. Characterization of microsomal and mitochondrial cyclophilin isoforms

The sequences of two cyclophilin (Cyp) isoforms from Dictyostelium discoideum have been determined. cyp2 is expressed as a 197 amino acid protein, which contains a 22 amino acid-long signal sequence, characteristic of endoplasmic reticulum localization signals, and that is cleaved in the mature protein. Mature Cyp2 has a molecular mass of 18 986 Da. The cyp3 gene encodes a 174 amino acid protein with a predicted molecular mass of 19 016 Da. Its sequence reveals no targeting sequence. From the MS analysis of affinity-purified cyclophilins from different subcellular compartments, we localized the previously described Cyp1 (Barisic K. et al., Dev. Genet. 12 (1991) 50-53) in cytosol, Cyp2 in microsomes and Cyp3 in mitochondria, respectively. The expression of cyp1 mRNA is constant during differentiation, whereas the mRNA level of both cyp2 and cyp3 is regulated and decreases steadily during development.     

Mutants of Dictyostelium discoideum blocked in expression of all members of the developmentally regulated discoidin multigene family

Mutant strains of D. discoideum are described that can complete morphogenesis and cytodifferentiation but which express vastly reduced levels of the galactose-binding lectins discoidin I and II (less than 1% and 1%-2% respectively) compared to the wild-type control. Mutant cells proceeding through development lack lectin activity, lectin protein, and specific lectin mRNA. In contrast, the genes encoding these proteins are present in their wild-type configurations in the genome. Since these proteins are encoded by four to five discrete genes, the mutations in these strains are most likely in genes involved in the regulation of the expression of members of this multigene family. The results also indicate that the discoidin lectins may not be required for fruiting body construction in this organism. Finally, coupled with the recent ability to transform D. discoideum, these mutants open the way to identification and isolation of regulatory genes and their products.     

Organization of the actin multigene family of Dictyostelium discoideum and analysis of variability in the protein coding regions

There are 17 to 20 actin genes in the genome of the cellular slime mold Dictyostelium discoideum. Genomic clones of 15 of the genes have been isolated. Extensive nucleotide sequence within the protein-coding regions has been determined, including the complete nucleotide sequence of four genes representing the three distinct evolutionary groups of Dictyostelium actin genes. All are similar to mammalian cytoplasmic actins at diagnostic amino acid positions, and there is generally less variability among Dictyostelium actin genes than among Drosophila actin genes. Two genes, Actins 3-sub 1 and 3-sub 2 differ substantially from all the rest in terms of replacement amino acid substitutions and probably encode actin-related proteins rather than bona fide actins. Each contains several amino acid substitutions that should alter the secondary structure of the resulting proteins, and Actin 3-sub 2 encodes four additional amino acids at the C terminus. This gene is as divergent from other Dictyostelium actin genes as is the yeast or a soybean actin gene. At present, evidence suggests that all 15 genes examined are expressed, except the previously identified Actin 2-sub 2. We suggest that Dictyostelium might maintain a high number of functional actin genes for the purpose of regulating the level of actin synthesis within narrow limits, rather than because most genes perform different functions.     

The wacA gene of Dictyostelium discoideum is a developmentally regulated member of the MIP family

We isolated a cDNA from Dictyostelium discoideum that encodes a 30 kDa protein with significant similarity to members of the major intrinsic protein (MIP) family of membrane transporters. The most closely related protein in the public data bases is an aquaporin from Cicadella viridis which shows 34% identity. The cDNA was used to isolate and characterize genomic fragments carrying the Dictyostelium gene which we named wacA. Genomic probes were used to recognize wacA mRNA isolated at various stages of development. The results showed that the gene is developmentally regulated such that the mRNA first appears at 12 h of development and is retained throughout the remainder of development. In situ hybridization of whole mounts prepared at 15 h of development showed that wacA mRNA accumulates exclusively in prespore cells and is absent from prestalk cells. Although wacA expression is prespore specific, disruption of the gene by homologous recombination did not result in observable alterations in the formation of spores or their resistance to osmotic challenges.     

A metabotropic glutamate receptor family gene in Dictyostelium discoideum

Metabotropic glutamate receptors (mGluRs) are a class of G-protein-coupled receptors that possess a seven transmembrane region involved in the modulation of excitatory synaptic transmission in the nervous system. mGluR orthologs have been identified in Drosophila, Caenorhabditis elegans, and higher organisms. Drosophila possesses two mGluR genes, DmGluRA and DmXR. We screened the Dictyostelium genome data base using the ligand binding domain of rat mGluR1 as bait, and identified a new receptor, DdmGluPR, belonging to the mGluR family. Similar to Drosophila DmXR, the residues of mGluRs involved in the binding of the alpha-carboxylic and alpha-amino groups of glutamate were well conserved in DdmGluPR, but the residues interacting with the gamma-carboxylic group of glutamate were not. The phylogenetic analysis suggests that DdmGluPR diverged after the mGluR family-GABA(B) receptors split but before mGluR family divergence. DdmGluPR mRNA was expressed in vegetative cells and throughout starvation-induced development, but the level of the expression was relatively high until 4 h after starvation. DdmGluPR was localized to the plasma membrane of axenically grown Ax-2 cells expressed as a green fluorescent protein fusion protein. DdmGluPR-null cells grew faster at high cell density and reached higher densities than wild-type cells. DdmGluPR-null cells exhibited delayed aggregates formation upon starvation and impaired chemotaxis toward cAMP. Although expressions of cAR1 and aca, cAMP-signaling components, were rapidly induced and peaked at 2-4 h in wild-type cells, DdmGluPR-null cells displayed sustained and peaked at 8 h of the expressions of these genes. Our findings suggest the involvement of DdmGluPR in the early development of Dictyostelium discoideum.     

Characterization of an antigenically related family of cell-type specific proteins implicated in slug migration in Dictyostelium discoideum

The monoclonal antibody MUD50 recognizes a group of developmentally regulated proteins, which are almost exclusively expressed by prespore cells in developing aggregates of Dictyostelium discoideum. Some of these antigens are integrally associated with the cell membrane, as assessed by physical and detergent-fractionation procedures. The MUD50-reactive proteins are glycosylated and some are phosphorylated. Post-translational modification is the common antigenic feature that is recognized by the MUD50 antibody in these cell-type-specific proteins. A glycosylation-defective mutant, DL118, (modB) does not express the MUD50 epitope, but does express the MUD52 epitope, which is found on a different group of glycoproteins. Therefore, we conclude that MUD50 recognizes a particular carbohydrate epitope on a restricted group of proteins. These proteins are structurally diverse, but are apparently involved in the maintenance of structure and movement of the multicellular D. discoideum slug.     

The Dictyostelium discoideum family of Rho-related proteins

Taking advantage of the ongoing Dictyostelium genome sequencing project, we have assembled >73 kb of genomic DNA in 15 contigs harbouring 15 genes and one pseudogene of Rho-related proteins. Comparison with EST sequences revealed that every gene is interrupted by at least one and up to four introns. For racC extensive alternative splicing was identified. Northern blot analysis showed that mRNAs for racA, racE, racG, racH and racI were present at all stages of development, whereas racJ and racL were expressed only at late stages. Amino acid sequences have been analysed in the context of Rho-related proteins of other organisms. Rac1a/1b/1c, RacF1/F2 and to a lesser extent RacB and the GTPase domain of RacA can be grouped in the Rac subfamily. None of the additional Dictyostelium Rho-related proteins belongs to any of the well-defined subfamilies, like Rac, Cdc42 or Rho. RacD and RacA are unique in that they lack the prenylation motif characteristic of Rho proteins. RacD possesses a 50 residue C-terminal extension and RacA a 400 residue C-terminal extension that contains a proline-rich region, two BTB domains and a novel C-terminal domain. We have also identified homologues for RacA in Drosophila and mammals, thus defining a new subfamily of Rho proteins, RhoBTB.     

Carbamoyl phosphate synthetase (CPSase) in the PYR1-3 multigene of Dictyostelium discoideum.

We have isolated and sequenced the genomic DNA from the slime-mould Dictyostelium discoideum multi-gene (PYR1-3) encoding the carbamoyl phosphate synthetase II domain (CPSase, EC.6.3.5.5). We describe sequencing by oligo-walking directly on PCR product in the solid-phase, avoiding subcloning procedures. The 2.4 kb fragment completes the sequence of the PYR1-3 gene, has no introns, and has the same structure as the rudimentary gene of Drosophila melanogaster. Comparison with the carbamoyl phosphate synthetases (CPSase I and CPSase II) of other species supports the hypothesis that this gene has arisen by tandem duplication from a smaller common ancestral gene in the progenote.     

A developmental shift in cell volume regulation in Dictyostelium discoideum

Abstract. We describe developmentally modulated volume regulation phenomena in Dictyostelium discoideum amebae. Transfer of cells to a medium of different osmolarity leads initially to volume shifts in the expected direction (cells shrink in concentrated media, swell in dilute media). Subsequently, the cells may slowly return to the original volume over 30–90 min. The capacity for volume regulation appears and disappears during development. A mutant isolated for lack of regulation shows abnormalities in developmental timing. Mutant enrichment according to volume regulatory properties may thus be useful in some lines of work.     

Changes in the Cell Surface Level of GP126 during Development of Dictyostelium discoideum

The developmental regulation of the vegetative cohesion molecule, gp 126, has been monitored in the cellular slime mould Dictyostelium discoideum. As judged by immunoprecipitation using an anti-vegetative cell, cohesion blocking antibody, gp126 persisted until at least the grex stage of development although a decline in the level of the molecule was observed thereafter. Further, after the grex stage, cells showed an increasing loss of ability to absorb the cohesion-blocking effect of an anti-vegetative cell Fab. Therefore, the decline of gp 126 could be ascribed to a loss from the cell surface. By radio-iodination of vegetative cells followed by liquid-scintillation counting of gp 126, developmental-regulation could be determined quantitatively.
At the grex stage of development, whole aggregates were embedded in wax. Longitudinal sections were then stained with a monospecific anti-gp 126 Fab, followed by a fluorescent sheep anti-rabbit IgG. Fluorescence was observed only at the tip (the prestalk) region, thereby showing that gp 126 is a prestalk marker.
To confirm the above result, grexes were dissociated and cells were separated into prespore and prestalk populations on a gradient of Percoll. Prestalk but not prespore cells were able to absorb the cohesion-blocking effect of an anti-vegetative cell Fab.
To examine the biosynthesis of gp 126, cells were pulsed with radioactive glucosamine, mannose or acetate. The pattern of incorporation of radioactivity suggested that the de novo synthesis of gp 126 ceases upon commencement of development.