Chromosomal mapping of tRNA genes from Dictyostelium discoideum

Summary Different wild-type isolates of Dictyostelium discoideum exhibit extensive polymorphism in the length of restriction fragments carrying tRNA genes. These size differences were used to study the organisation of two tRNA gene families which encode a tRNA^Val(GUU) and a tRNA^Val(GUA) gene. The method used involved a combination of classitics. The tRNA genes were mapped to specific linkage groups (chromosomes) by correlating the presence of polymorphic DNA bands that hybridized with the tRNA gene probes with the presence of genetic markers for those linkage groups. These analyses established that both of the tRNA gene families are dispersed among sites on several of the chromosomes. Information of nine tRNA^Val(GUU) genes from the wild-type isolate NC4 was obtained: three map to linkage group I (C, E, F,), two map to linkage group II (D, I), one maps to linkage group IV (G), one, which corresponds to the cloned gene, maps to either linkage group III or VI (B), and two map to one of linkage groups III, VI or VIII (A, H). Six tRNA^Val(GUA) genes from the NC4 isolate were mapped; one to linkage group I (D), two to linkage group III, VI or VII (B, C) and three to linkage group VII or III (A, E, F).     

Ras-related genes in Dictyostelium discoideum

Dictyostelium discoideum, like other eukaryotes, has been shown to express several ras-related genes. Two gene products, Ddras and DdrasG, are highly conserved relative to the human ras proteins. Ddras is expressed at the pseudoplasmodial stage of development, whereas DdrasG is expressed in vegetative cells and during early development. In addition, Dictyostelium possesses three ras-related genes, SAS1, SAS2 and Ddrap1, whose gene products are only partially conserved relative to those of the ras genes. The expression of these three genes is also developmentally regulated.     

Primary structure and regulation of vegetative specific genes of Dictyostelium discoideum

We have examined the expression and structure of several genes belonging to two classes of vegetative specific genes of the simple eukaryote, Dictyostelium discoideum. In amebae grown on bacteria, deactivation of all vegetative specific genes occurred at the onset of development and very little mRNA exists by 8 to 10 hours. In contrast, when cells were grown in axenic broth, the mRNA levels remained constant until a dramatic drop occurred around 10 to 12 hours. Thus, regulation of both classes of genes during the first several hours of development is dependent upon the prior growth conditions. Analysis of genomic clones has resulted in the identification of two V genes, V1 and V18, as ribosomal protein genes. Several other V genes were not found to be ribosomal protein genes, suggesting that in Dictyostelium non-ribosomal protein genes may be coordinately regulated with the ribosomal protein genes. Finally, using deletion analysis we show that the promoters of two of the V genes are composed of a constitutive positive element(s) located upstream of sequences involved in the regulated expression of these genes and within the first 545 upstream bp for V18 and 850 bp for V14. The regions involved in regulated expression were localized between -7 and -222 for V18 and -70 and -368 for V14. The sequences conferring protein synthesis sensitivity were shown to reside between -502 and -61 of the H4 promoter.     

Developmentally regulated protein-tyrosine kinase genes in Dictyostelium discoideum.

Dictyostelium discoideum, an organism that undergoes development and that is amenable to biochemical and molecular genetic approaches, is an attractive model organism with which to study the role of tyrosine phosphorylation in cell-cell communication. We report the presence of protein-tyrosine kinase genes in D. discoideum. Screening of a Dictyostelium cDNA expression library with an anti-phosphotyrosine antibody identifies fusion proteins that exhibit protein-tyrosine kinase activity. Two distinct cDNAs were identified and isolated. Though highly homologous to protein kinases in general, these kinases do not exhibit many of the hallmarks of protein-tyrosine kinases of higher eucaryotes. In addition, these genes are developmentally regulated, which suggests a role for tyrosine phosphorylation in controlling Dictyostelium development.     

Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes.

A previously isolated cDNA clone, pLK229, that is specific for mRNA developmentally expressed during Dictyostelium discoideum spore germination and multicellular development, was used to screen two genomic libraries. Two genomic sequences homologous to pLK229 were isolated and sequenced. Genomic clone p229 is identical to the cDNA clone pLK229 and codes for a polypeptide of 381 amino acids. This polypeptide is composed of five tandem repeats of the same 76-amino-acid sequence. Clone lambda 229 codes for a protein of 229 amino acids, containing three tandem repeats of the identical 76-amino-acid sequence. A computer search for homology to known proteins revealed that the 76-amino-acid repeat was identical to human and bovine ubiquitin except for two amino acid differences.     

Genetic and cytological characterisation of fusion chromosomes of Dictyostelium discoideum

Abnormally large chromosomes which appear to result from the fusion of 2 chromosomes of the normal karyotype have been found in diploids of Dictyostelium discoideum formed by parasexual fusion of haploid strains HU483 (n=7) and HU245 (n=7). These fusion chromosomes appear to be the products of the tandem translocation of most, if not all, of one acrocentric chromosome to the telomere of a second acrocentric. Thus the chromosome number of the diploids is reduced from the normal 2n=14 to 2n=13 with the formation of an abnormally large acrocentric fusion chromosome. Experimental haploidisation of such diploids results in two types of products, those with a normal 7 chromosome karyotype and those with an abnormal 6 chromosome karyotype which contains the fusion chromosome. Genetic analysis of haploid segregants indicates that linkage groups II and VII are involved in this fusion. Phenotypes of recombinant diploids obtained following mitotic crossing-over establishes that linkage group II is proximal to linkage group VII. Cytological examination of the karyotypes of haploid strains bearing the fusion chromosome suggest that chromosome 2 may correspond to linkage group II and chromosome 3 to linkage group VII. Haploid strains bearing the fusion chromosome grow and develop normally so little or no genetic information can have been lost in the fusion event. While the nature of this event is unknown it may have involved aberrant recombinational DNA repair since the parental haploid strain HU483 bears the radB13 DNA repair mutation.     

Monoclonal antibodies specific for stalk differentiation in Dictyostelium discoideum

We have produced two monoclonal antibodies specific to the stalk cells of Dictyostelium discoideum fruiting bodies. Both monoclonal antibodies react with high molecular weight proteins previously found to be stalk-specific by two-dimensional gel analysis. One antibody (JAb 1) is specific for a single protein of apparent molecular weight 310 000 which first appears when overt stalk differentiation begins at 20 h. The other monoclonal antibody (JAb 2) is also stalk-specific, though earlier in development it binds to proteins extracted from both prestalk and prespore cells of the migrating slug. It reacts with two proteins in stalks, one of apparent molecular weight 430 000 which is first detected during tip formation at 12 h and a lower molecular weight protein (310 000) detected from 20 h. Although several markers are available for the investigation of prespore/spore differentiation there is a distinct lack of suitable prestalk/stalk markers. The monoclonal antibodies described here are highly specific stalk markers and should prove useful in the study of cell proportioning and terminal differentiation.     

Control of N-acetylglucosaminidase specific activity in myxamoebae of Dictyostelium discoideum

Myxamoebae of Dictyostelium discoideum were grown with Aerobacter aerogenes as substrate, on nutrient agar plates or in shaken culture, with mean doubling times that varied between 3 and 6 hr. Growth in axenic culture was with mean doubling times of 5–6 or 8 hr. The specific activity of N-acetylglucosaminidase was three to four times higher in myxamoebae grown axenically than in myxamoebae grown with A. aerogenes and there was no correlation between enzyme specific activity and myxamoebal growth rate. High specific activities of N-acetylglucosaminidase were also found in myxamoebae grown with gram-positive bacteria (Bacillus megaterium, Staphylococcus lactis) as substrate while low specific activities were found in myxamoebae grown with the gram-negative bacteria Acinetobacter lwoffi and Pseudomonas aeruginosa as well as with A. aerogenes. A preparation of the cell envelope of A. aerogenes was nearly as effective as the intact bacteria at depressing myxamoebal N-acetylglucosaminidase specific activity. Lipopolysaccharide extracted from the cell envelope of gram-negative bacteria, or the lipid A component of the lipopolysaccharide, also depressed N-acetylglucosaminidase specific activity when added to axenic cultures of myxamoebae.     

Synthesis of specific proteins for sexual development in Dictyostelium discoideum

The development of Dictyostelium discoideum may proceed by two pathways, macrocyst or fruiting-body formation, the former being the sexual and the latter the asexual cycle. The pathway of development depends on the presence or absence of zygote giant cells which are produced through fusion of opposite mating-type cells in a population, in heterothallic strains. During the early stages of macrocyst development the patterns of developmentally regulated proteins were noted to differ considerably from those during fruiting-body development. Furthermore, the haploid cells around zygote giant cells synthesized a large number of specific proteins for macrocyst development through the influence of giant cells.     

Phototaxis genes on linkage group V in Dictyostelium discoideum

Abstract During the course of mapping and complementation analysis of phototaxis ( pho ) mutations in Dictyostelium discoideum we have assigned to linkage group V three mutant pho alleles belonging to complementation groups phoG and phoK . These are the first genetic markers with an easily recognizable phenotype to be found on this linkage group.     

Identification and characterization of two flavohemoglobin genes in Dictyostelium discoideum

Flavohemoglobins are being identified in an expanding number of prokaryotes and unicellular eukaryotes. These molecules consist of an N-terminal hemoglobin domain and a C-terminal oxidoreductase domain, and are considered to function in storage or as sensors for O2, and in defense against oxidative stress and/or NO. However, their physiological significance has not yet been determined. Here, we isolated and analyzed two flavohemoglobin genes of Dictyostelium discoideum, DdFHa and DdFHb, which lie close to each other in the genome. DdFHs were induced by submerged conditions, and enriched in the sexually mature cells of D. discoideum. Although they were not essential for growth or development under standard laboratory conditions, disruption of both genes caused an increase in number of large but uninuclear cells, and hypersensitivity to higher concentrations of glucose and to NO releasers. These results indicate that DdFHs are responsible for transducing NO signals to maintain normal cellular conditions against environmental stresses.     

Codon preference in Dictyostelium discoideum.

Dictyostelium discoideum is of increasing interest as a model eukaryotic cell because its many attributes have recently been expanded to include improved genetic and biochemical manipulability. The ability to transform Dictyostelium using drug resistance as a selectable marker (1) and to gene target by high frequency homologous integration (2) makes this organism particularly useful for molecular genetic approaches to cell structure and function. Given this background, it becomes important to analyze the codon preference used in this organism. Dictyostelium displays a strong and unique overall codon preference. This preference varies between different coding regions and even varies between coding regions from the same gene family. The degree of codon preference may be correlated with expression levels but not with the developmental time of expression of the gene product. The strong codon preference can be applied to identify coding regions in Dictyostelium DNA and aid in the design of oligonucleotide probes for cloning Dictyostelium genes.     

Developmental decisions in Dictyostelium discoideum.

A few hours after the onset of starvation, amoebae of Dictyostelium discoideum start to form multicellular aggregates by chemotaxis to centers that emit periodic cyclic AMP signals. There are two major developmental decisions: first, the aggregates either construct fruiting bodies directly, in a process known as culmination, or they migrate for a period as "slugs." Second, the amoebae differentiate into either prestalk or prespore cells. These are at first randomly distributed within aggregates and then sort out from each other to form polarized structures with the prestalk cells at the apex, before eventually maturing into the stalk cells and spores of fruiting bodies. Developmental gene expression seems to be driven primarily by cyclic AMP signaling between cells, and this review summarizes what is known of the cyclic AMP-based signaling mechanism and of the signal transduction pathways leading from cell surface cyclic AMP receptors to gene expression. Current understanding of the factors controlling the two major developmental choices is emphasized. The weak base ammonia appears to play a key role in preventing culmination by inhibiting activation of cyclic AMP-dependent protein kinase, whereas the prestalk cell-inducing factor DIF-1 is central to the choice of cell differentiation pathway. The mode of action of DIF-1 and of ammonia in the developmental choices is discussed.     

Phosphotyrosine-containing proteins in Dictyostelium discoideum.

Phosphotyrosine-containing proteins in Dictyostelium discoideum were detected by immunoblot analysis and immunoprecipitation using a monoclonal anti-phosphotyrosine antibody. The iodinated antibody recognized on bots a cluster of 205-220 kDa polypeptides and bands of 107 and 60 kDa. The 107 and 60 kDa polypeptides and, in addition, a 82 kDa one became phosphorylated on tyrosine when the immunoprecipitate was incubated with [gamma-32P]ATP. In preparations from differentiating cells the intensity of the label was increased in the 60 kDa band and decreased in the 107 and 205-220 kDa bands.     

Spore coat genes SP60 and SP70 of Dictyostelium discoideum.

We cloned and sequenced the genes for two of the major proteins found in spore coats of Dictyostelium discoideum. The predicted translation product of each of these genes starts with a hydrophobic signal sequence that is subsequently cleaved. Expression of these spore coat genes is coordinate in prespore cells.