The 3'-noncoding region of the chick myosin light-chain gene hybridizes to a family of repetitive sequences in the slime mold Dictyostelium discoideum

During studies aimed at isolating myosin-specific genomic clones in Dictyostelium, we probed a lambda genomic library with a chicken myosin light-chain sequence (pML10). Many lambda recombinant Dictyostelium clones hybridized to the pML10 cDNA insert, indicating that this sequence was reiterated in the Dictyostelium genome. It was found that the 3'-noncoding region (pML10-NC) alone was responsible for these results. Dictyostelium DNA contained approximately 65 copies of a sequence(s) similar but not identical to that of pML10-NC. Southern blot analysis showed that pML10-NC hybridized to many Dictyostelium genomic DNA fragments of varying sizes generated by digestion with EcoRI, HindIII, or AluI. In addition, each of the Dictyostelium clones was different in its size, restriction map, and flanking sequences. It seems likely, therefore, that the sequences which hybridized to pML10-NC are scattered throughout the Dictyostelium genome and similar but not identical to each other or to pML10-NC. Thus, probing with pML10-NC has allowed us to select a family of closely related but not identical sequences. These D. discoideum sequences are not found in other slime mold species. No RNA complementary to pML10-NC was found in vegetative cells, 18 h culmination stage, spores, or 1- and 2-h germinating spores. pML10-NC-related sequences were present in two other Dictyostelium species but were absent in the related genus Polysphondylium.     

Correlation between acrasins and spore germination inhibitors in cellular slime molds.

Discadenine,3-(3-amino-3-carboxypropyl)-N6-delta 2-isopentenyladenine, which inhibits spore germination, was previously found in Dictyostelium discoideum. Studies on the distribution of discadenine in different species of cellular slime molds by high-pressure liquid chromatography showed that discadenine is present in D. discoideum, Dictyostelium purpureum, and Dictyostelium mucoroides, but not in Dictyostelium minutum, Polysphondylium violaceum, or Polysphondylium pallidum. Discadenine synthetase, which is involved in biosynthesis of discadenine with N6-delta 2-isopentenyladenine as substrate, was only detected in cells of the former three species. In addition, discadenine inhibited spore germination only in these three species. These results clearly demonstrate that discadenine is produced as an inhibitor of spore germination in the species of cellular slime molds in which the acrasin is cyclic adenosine 5'-monophosphate (AMP). This means that there is a structural and biochemical correlation between the spore germination inhibitor and the acrasin, since 5'-AMP, a direct precursor in discadenine biosynthesis, can be derived from cyclic AMP by hydrolysis with cyclic AMP phosphodiesterase.     

Occurrence and distribution of dictyostelid cellular slime molds in Norway

The distribution of eleven dictyostelid cellular slime molds found in Norway was studied. They were divided into four groups according to their dominance. In decreasing order the groups were: (1) two varieties of Dictyostelium mucoroides , (2) D. aureostipes and Polysphondylium violaceum , (3) D. fasciculatum, D. minutum, P. pallidum and Dictyostelium sp. 1, and (4) Dictyostelium sp. 2 and 3 and Acytostelium lep–tosomum. Differences in distribution related to altitudes and latitudes, climates and dominant tree species were observed for some species.     

Characterization of cDNA clones specific for sequences developmentally regulated during Dictyostelium discoideum spore germination.

Spore germination in the slime mold Dictyostelium discoideum was used as a model to study the developmental regulation of protein and mRNA synthesis. Changes in the synthesis of these macromolecules occur during the transition from dormant spore to amoebae. The study of the mechanisms which regulate the quantity and quality of protein synthesis can best be accomplished with cloned genes. cDNA clones which hybridized primarily with mRNAs from only spores or germinating spores and not with growing amoebae were collected. Three such clones, denoted pLK109, pLK229, and pRK270, were isolated and had inserts of approximately 500, 1,200, and 690 base pairs, respectively. Southern blot hybridization experiments suggested that each of the genes is present in multiple copies in the D. discoideum genome. RNA blot hybridizations were performed to determine the sizes of the respective mRNAs and their developmental regulation. The mRNA that hybridized to pLK109 DNA was present predominantly in spores and at 1 h after germination but was absent in growing amoebae. Its concentration dramatically dropped at 3 h. The mRNA present in spores is apparently larger (approximately 0.5 kilobase) than in the later stages of germination (0.4 kilobase), indicating processing of the RNA during germination. The mRNA that hybridized to pLK229 DNA was approximately 1.0 kilobase and was present in very low amounts during growth. Its concentration rose until 1 h after spore germination and decreased thereafter. pRK270-specific RNA was approximately 2.7 kilobases and was found predominantly at 1 h after germination. It was present in lower concentrations at 2 and 3 h after germination and was absent in spores and amoebae. In vitro translation of mRNA selected from 1-h polyadenylated RNA which was hybridized to pLK109 or pLK229 DNA gave proteins of molecular weights consistent with the sizes of the mRNAs as determined by the RNA blot analysis.     

Dictyostelids living in the soils of the Atlantic forest, Iguazú region, Misiones, Argentina: description of new species

Thirteen new species and varieties of dictyostelid cellular slime molds (csm) were isolated from soils of the Atlantic Subtropical Rain Forest at the Iguazú Falls, Northeastern Misiones Province, Argentina. Seven new species are described herein, one of them is a Polysphondylium, while the rest of the species belong to the genus Dictyostelium. Also, six taxa are new varieties of Dictyostelium and Acytostelium, which will be reported later. Fourteen Northern Hemisphere (Tikal) species have also been isolated from Iguazú soils, some of them new records for Southern South America. This csm community, when compared with others from forests of the Northern Hemisphere, particularly Tikal, Guatemala, give some insight into a possibly different evolutionary history and/or natural selection in the two areas.     

Isolation, characterization, and analysis of Leymus-specific DNA sequences.

Genomic Southern hybridization using labeled total genomic DNA of Leymus mollis as probe showed intense hybridization signals on all restriction enzyme digested DNA from five species of Leymus Hochst., and four species of Psathyrostachys Nevski. Experiments using the same L. mollis probe, but with unlabeled blocking DNA from Psathyrostachys, showed no hybridization at all. These two genera evidently had the same genomic content. Southern hybridization without blocking allowed identification of DNA fragments abundant in Leymus and Psathyrostachys. Fragments potentially specific to Leymus were cloned. Five repetitive DNA clones from L. mollis and L. arenarius were characterized: pLmIs1, pLmIs44, pLmIs51, pLmIs53, and pLaIs56. These clones hybridized to both Leymus and Psathyrostachys on Southern blots - no clone hybridized to only one of these genera. Both Southern blot and fluorescence in situ hybridization (FISH) experiments showed that all the clones contained dispersed repetitive sequences. They painted all and whole chromosomes uniformly except at centromeres, telomeres, and nucleolar organiser regions. Three of these clones, i.e., pLmIs1, pLmIs44, and pLmIs53, were essentially specific to Leymus and Psathyrostachys - little or no hybridization was detected in other genera such as Triticum, Hordeum, Thinopyrum, or Elymus. Sequence analysis further revealed that the clones were part of retroelements. In particular, the clone pLmIs44 produced hybridization profiles suitable for analysis of genetic relatedness among species. The present study shows that Leymus and Psathyrostachys share the same basic genome, Ns, and therefore provides strong evidence for combining these two genera.     

CELLULAR SLIME MOLDS OF SWITZERLAND. I. DESCRIPTION OF NEW SPECIES

During the study of the distribution of cellular slime molds in Swiss forest soils, three new species were isolated and studied in culture, namely, Dictyostelium fasciculatum, Dictyostelium polycarpum and Polysphondylium filamentosum. The first is cosmopolitan, the latter two are known only from Switzerland. Dictyostelium polycarpum is a very delicate species and is the first species reported which is restricted to a high altitude environment. It is characterized by a coremiform habit, reminiscent of D. polycephalum, and relatively long reniform spores. Dictyostelium fasciculatum is a larger species which resembles D. mucoroides in gross morphology but is much closer to Polysphondylium in its life cycle pattern and behavior. Polysphondylium filamentosum is only the third well-defined species of the genus to be described. It produces filamentose principal and lateral axes. Optimum temperature for all three is around 20 C, somewhat below that of most other species in the family.     

The simple repeat poly(dT-dG).poly(dC-dA) common to eukaryotes is absent from eubacteria and archaebacteria and rare in protozoans

Genomic DNA from a wide variety of prokaryotic and eukaryotic organisms has been assayed for the simple repeat sequence poly(dT-dG).poly(dC-dA) by Southern blotting and DNA slot blot hybridizations. Consistent with findings of others, we have found the simple alternating sequence to be present in multiple copies in all organisms in the animal kingdom (e.g., mammals, reptiles, amphibians, fish, crustaceans, insects, jellyfish, nematodes). The TG element was also found in lower eukaryotes (Saccharomyces cerevisiae, Neurospora crassa, and Dictyostelium discoideum) and at a much lower frequency in protozoans (Oxytricha fallux and Tetrahymena thermophila). The sequence was also repeated in high copy number in a higher plant (Zea mays) as well as at very high levels in a unicellular green alga (Chlamydomonas reinhardi). Although the copy number of the repeat per haploid genome was generally proportional to genome size, there was a greater-than-1,000-fold variation in the number of (TG)25/100-kb genomic DNA. By contrast, no eu-or archaebacterium--including Myxococcus xanthus, whose life cycle is very similar to that of the slime mold Dictyostelium discoideum, and Halobacter volcanii, whose genome contains other repeated sequences-- was found whose genomic DNA contained this sequence in detectable amounts. A computer search also failed to find the TG element in human mitochondrial DNA.      

CELLULAR SLIME MOLDS OF SOUTHEAST ASIA. I. DESCRIPTION OF NEW SPECIES

Sixteen species of cellular slime molds were isolated from Southeast Asian forest soils. Ten of these, Dictyostelium mucoroides Brefeld, D. purpureum Olive, D. polycephalum Raper, D. lacteum van Tieghem, D. rhizopodium Raper and Fennell, D. lavandulum Raper and Fennell, D. vinaceo-fuscum Raper and Fennell, D. coeruleo-stipes Raper and Fennell, Polysphondylium violaceum Brefeld, and P. pallidum Olive have been previously described and are well-recognized species occurring in other parts of the world. Two, one in the genus Dictyostelium and one belonging to the family Guttulinaceae, are considered species by the author but have not been formally described. Four are described in this paper as new: Dictyostelium intermedium, D. multi-stipes, D. bifurcatum, and Acytostelium subglobosum. A new variety papilloideum of D. lacteum is also described. One other discovery of special interest is an isolate of Polysphondylium violaceum which produces abundant macrocysts, now known to be the sexual stage in the life cycle of cellular slime molds.     

Characterization of DNA Sequences Cloned From Resting Spores of Plasmodiophora brassicae

Forty-four clones containing P. brassicae genomic DNA fragments were isolated from a partial genomic library (pUC118) of the fungus. Eleven DNA fragments were arbitrarily selected from them and partially sequenced. Southern blot analysis was performed using the 11 DNA fragments as probes to estimate the copy number of these DNA fragments in the P. brassicae genome and to see if they hybridized also to genomic DNA of host plant (Chinese cabbage). Five of those (Nos 24, 48, 54, 57, and 108) were of single-copy and hybridized only to DNA bands of P. brassicae. Two fragments (Nos 4 and 15) were found to contain repetitive sequence of/", brassicae. Three fragments (Nos 69,73, and 82) hybridized to DNA bands of both host and P. brassicae while the size and the number of the bands were different between host and P. brassicae. Data base searches revealed that one of the single-copy fragments (No. 48) had high homology with yeast RNA polymerase II gene.     

Intracellular cyclic GMP-binding proteins in cellular slime molds.

The complexity of cyclic GMP-binding activity in the 48,000 X g supernatant of three species of the cellular slime molds (Dictyostelium discoideum, Dictyostelium rosarium, and Polysphondylium violaceum) was studied by gel filtration chromatography on AcA 34 Ultrogel. All these species have in common a cyclic GMP-binding protein of molecular weight of about 2.5 X 10(5) which specifically binds this nucleotide. In addition, Scatchard plots of assays carried out with the 48,000 X g supernatant of these species exhibit cyclic GMP-binding activity with an apparent dissociation constant of about 1 nM. None of the cyclic GMP-binding proteins separated by chromatography on AcA 34 Ultrogel was associated to protein kinase activity stimulation. In view of the cyclic GMP function during chemotactic transduction in the cellular slime molds, the possible molecular function for this 2.5 X 10(5)-dalton cyclic GMP-binding protein is discussed.     

Dictyostelid cellular slime molds associated with grasslands of the central and western United States

Dictyostelid cellular slime molds (dictyostelids) associated with grassland ecosystems of the central and western United States were investigated at nine sites that included examples of the three major ecological types of grasslands (tall grass, mixed grass and short grass) generally recognized for the region. Samples of soil/humus collected from each site were examined with the Cavender method of isolating dictyostelids. For each of those six sites with well developed gallery forests present, an additional set of forest soil/humus samples was collected. A more intensive sampling effort was carried out at one site (Konza LTER) to assess the possible effects of burning and grazing on dictyostelid diversity and density. Twelve species of dictyostelids were recovered from grassland sites, whereas gallery forest sites yielded only nine species. Four cosmopolitan species (Dictyostelium giganteum, D. mucoroides, D. sphaerocephalum and Polysphondylium pallidum) were represented by the greatest densities of clones, with D. sphaerocephalum particularly common. The general pattern across all sites was that both species richness and density of dictyostelids decreased with decreasing precipitation. Samples collected from ungrazed grassland plots yielded higher numbers of both species and clones as compared to grazed plots, and the general pattern was for both values to increase as the interval between fires increased. For numbers of clones this correlation was statistically significant.     

Isolation and characterization of serine protease gene(s) from Perkinsus marinus

This study reports the first serine protease gene(s) isolated from Perkinsus marinus. Using universal primers, a 518 bp subtilisin-like serine protease gene fragment was amplified from P. marinus genomic DNA and used as a probe to screen a lambda-phage P. marinus genomic library; 2 different lambda-phage clones hybridized to the digoxigenin(DIG)-labeled subtilisin-like gene fragment. Following subcloning and sequencing of the larger DNA fragment, a 1254 bp open reading frame was identified and later confirmed, by 5' and 3' random amplification of cDNA ends (RACE) and northern blot analysis, to contain the entire coding-region sequence. Sequence analysis of the 3' RACE results from 2 isolate cultures, VA-2 (P-1) and LA 10-1, revealed multiple polymorphic sites within and among isolates. We identified 2 different types of cDNA clones with 95.53% nucleotide sequence similarity, suggesting the possibility of 2 closely related genes within the P. marinus genome. Southern blot analysis of genomic DNA from 12 genetically distinct P. marinus isolate cultures revealed 2 different banding patterns among isolates.     

New species of dictyostelids from Patagonia and Tierra del Fuego, Argentina

In late Jan and early Feb 2005 samples for isolation of dictyostelid cellular slime molds (dictyostelids) were collected in five different provinces and from six national parks (all located 39-55°S) in Patagonia and Tierra del Fuego, Argentina. Southern beech (Nothofagus) forests represented the primary vegetation type investigated, but some samples were obtained from Patagonian steppe, alpine meadows, Valdivian temperate rainforests and coniferous forests dominated by Araucaria, Austrocedrus and Fitzroya. Among the dictyostelids isolated from the samples we collected were seven species new to science. These species (Dictyostelium austroandinum, D. chordatum, D. fasciculoideum, D. gargantuum, D. leptosomopsis, D. valdivianum and Polysphondylium patagonicum) are described herein on the basis of both morphology and molecular (SSU rDNA) data. One of the new species, D. gargantuum, is one of the largest representatives of the group reported to date. Another unusual species, D. chordatum, produces long interwoven sorocarps that do not appear to respond to a spacing gas similar to the condition first noted in D. implicatum.     

The influence of light and different ATP concentrations on cell aggregation in cyclic AMP sensitive and insensitive species of the cellular slime molds

The influence of light and different concentrations of ATP on cell aggregation in cyclic AMP sensitive (Dictyostelium mucoroides, D. purpureum) and cyclic AMP insensitive species (Polysphondylium violaceum, P. pallidum, D. lacteum) of the cellular slime molds was observed in small and in large amoebal populations.Both light and ATP (optimal concentration:10^-5M) accelerated cell aggregation and increased the number of aggregating centers in large populations. For cyclic AMP sensitive species the effect of ATP in large populations was more pronounced than for the species that do not react to cyclic AMP.A possible explanation for the similar effect of light and ATP has been discussed.     

Mitochondrial Ribosomal Protein L11 Gene of Dictyostelium discoideum Resides not in the Nuclear Genome but in the Mitochondrial Genome

During the course of analysis of the mitochondrial genome ofthe cellular slime mold, Dictyostelium discoideum, we founda gene (rpl11) for mitochondrial ribosomal protein L11 (RPL11),having 172 amino acid residues. Southern blot analysis revealedthat the gene resided in the mitochondrial DNA as a singlecopybut not in the nuclear DNA. From Northern blot experiments,one major mRNA (about 27 kb) and two minor mRNAs (about 4 and5 kb) for the gene were detected in the mitochondria. This isthe first report showing that the active gene for RPL11 stillresides in the mitochondrial genome and has not been transferredto the nuclear genome in D. discoideum.     

Evolutionary conservation of the human 7 S RNA sequences

The cloning and characterization of the cytoplasmic 7 S RNAs of HeLa cells has provided pure probes to study the organization of the corresponding genomic DNA sequences. Such analysis has shown that the 7 S L and K RNAs are derived from families of middle repetitive DNA (Ullu & Melli, 1982; Ullu et al., 1982). In this work we analyze the evolutionary conservation of these sequences in the RNA and DNA of distantly related species. Hybridization of the 7 S recombinants to the RNA of rodents, birds, amphibians and echinoderms suggests high conservation of these sequences throughout evolution. Southern blot analysis of genomic DNAs from the same species shows the presence of families of repeated sequences homologous to the 7 S recombinants and Alu DNAs in the genomes of the same species. We were unable to hybridize the 7 S probes to the RNAs of Drosophila melanogaster or Dictyostelium discoideum, although sequence(s) homologous to the 7 S L probe were found in the genome of D. discoideum and to both 7 S L and K probes in the genome of D. melanogaster.     

Alpha-actinin synthesis can be modulated by antisense probes and is autoregulated in non-muscle cells.

We used a 279 bp cDNA probe derived from a Dictyostelium alpha-actinin genomic sequence to assay the degree of homology between alpha-actinin from slime molds, mammalian and chicken cells. Recognition of this probe by vertebrate cells was shown in Southern and Northern blots, and by antisense RNA-induced depression of endogenous alpha-actinin synthesis in living cells. Micro-injection of Dictyostelium or chicken gizzard alpha-actinin resulted in incorporation of these proteins in stress fibers, peripheral microfilament belts and adhesion sites. Alpha-actinin-injected cells showed a marked, transient reduction of synthesis of the corresponding endogenous protein. These data emphasize the high degree of conservation of alpha-actinin during evolution and show for the first time autoregulation of synthesis for a microfilament protein.     

The regulation of cellular slime mold development: a factor causing development of Polysphondylium violaceum aggregation-defective mutants.

aggA mutants of Polysphondylium violaceum develop normally in synergistic mixtures with other aggregation-defective mutants. Cell to cell contact is not necessary for development. A small dialyzable factor(s) produced by wild-type and other aggregation-defective mutants triggers development of aggA mutants. This factor (D factor) is developmentally regulated, appearing early in development and then disappearing. Mutants require D factor until aggregation has just begun and then they can continue even in the absence of added factor. D factor is produced by many, but not all species of cellular slime molds and is developmentally regulated in Dictyostelium discoideum as well as P. violaceum.