Dictyostelium Discoideum NC-4 Research Articles

Internally Located and Oppositely Oriented Polymerase II Promoters Direct Convergent Transcription of a LINE-Like Retroelement, the Dictyostelium Repetitive Element, from Dictyostelium discoideum

The Dictyostelium discoideum NC4 genome harbors approximately 150 individual copies of a retrotransposable element called the Dictyostelium repetitive element (DRE). This element contains nonidentical terminal repeats (TRs) consisting of conserved building blocks A and B in the left TR and B and C in the right TR. Seven different-sized classes of RNA transcripts from these elements were resolved by Northern (RNA) blot analysis, but their combined abundance was very low. When D. discoideum cells were grown in the presence of the respiratory chain blocker antimycin A, steady-state concentrations of these RNA species increased 10- to 20-fold. The D. discoideum genome contains two DRE subtypes, the full-length 5.7-kb DREa and the internally deleted 2.4-kb DREb. Both subtypes are transcribed, as confirmed by analysis of cloned cDNA. Primary transcripts from the sense strand originate at nucleotide +1 and terminate at two dominant sites, located 21 or 28 nucleotides upstream from the 3' end of the elements. The activity of a reasonably strong polymerase II promoter in the 5'-terminal A module is slightly upregulated by the tRNA gene located 50 +/- 4 nucleotides upstream and drastically reduced by the adjacent B module of the DRE. Transcripts from the opposite DNA strand (complementary-sense transcripts) were also detected, directed by an internally located polymerase II promoter residing within the C module. This latter transcription was initiated at multiple sites within the oligo(dA12) stretch which terminates DREs.

Internally located and oppositely oriented polymerase II promoters direct convergent transcription of a LINE-like retroelement, the Dictyostelium repetitive element, from Dictyostelium discoideum.

The Dictyostelium discoideum NC4 genome harbors approximately 150 individual copies of a retrotransposable element called the Dictyostelium repetitive element (DRE). This element contains nonidentical terminal repeats (TRs) consisting of conserved building blocks A and B in the left TR and B and C in the right TR. Seven different-sized classes of RNA transcripts from these elements were resolved by Northern (RNA) blot analysis, but their combined abundance was very low. When D. discoideum cells were grown in the presence of the respiratory chain blocker antimycin A, steady-state concentrations of these RNA species increased 10- to 20-fold. The D. discoideum genome contains two DRE subtypes, the full-length 5.7-kb DREa and the internally deleted 2.4-kb DREb. Both subtypes are transcribed, as confirmed by analysis of cloned cDNA. Primary transcripts from the sense strand originate at nucleotide +1 and terminate at two dominant sites, located 21 or 28 nucleotides upstream from the 3' end of the elements. The activity of a reasonably strong polymerase II promoter in the 5'-terminal A module is slightly upregulated by the tRNA gene located 50 +/- 4 nucleotides upstream and drastically reduced by the adjacent B module of the DRE. Transcripts from the opposite DNA strand (complementary-sense transcripts) were also detected, directed by an internally located polymerase II promoter residing within the C module. This latter transcription was initiated at multiple sites within the oligo(dA12) stretch which terminates DREs.

Efficient Induction of Sporulation of Dictyostelium Prespore Cells by 8-Bromocyclic AMP under Both Submerged- and Shaken-Culture Conditions and Involvement of Protein Kinase(s) in Its Action.

It is important to establish an experimental system in which sporulation of Dictyostelium can be induced at high cell densities to obtain sufficient amounts of materials for analysis of the molecular events leading to sporulation. 8-Bromo cAMP (Br-cAMP) was found to be effective for inducing sporulation by prespore cells of Dictyostelium discoideum NC4 at high cell densities under both submerged- and shaken-culture conditions. Ultrastructural studies revealed that the morphological changes associated with this sporulation proceeded normally in vitro. The effect of Br-cAMP was inhibited by two protein kinase inhibitors, K252a and staurosporine. Protein-phosphorylation experiments showed that Br-cAMP induced increased phosphorylations of a 96 kDa spore coat protein (SP96) and a protein with a mobility corresponding to a molecular weight of 50 kDa (p50-4). The protein kinase inhibitor K252a blocked the phosphorylations of both proteins. These proteins may be targets of particular protein kinase(s) that is activated by Br-cAMP. These findings indicate that the present experimental system should be useful for elucidating the molecular events involved in normal sporulation and the mechanism by which Br-cAMP induces sporulation in vitro.

RNA synthesis during germination of UV-irradiated Dictyostelium discoideum spores.

UV irradiation to the spores of Dictyostelium discoideum NC4 resulted in a more prolonged delay of amoeba-emergence from swollen spores with increasing UV fluence. During the germination, an inhibition of total RNA synthesis and a shift of stage of maximum RNA synthesis to the later period were observed. The maximum poly(A)+ RNA synthetic activity was found on an early stage of amoeba-emergence prior about 1 h to the beginning of rRNA synthesis in unirradiated spore germination; but, in UV-irradiated spore germination, the stage of maximum poly(A)+ RNA synthesis shifted to the later stage of germination with increasing UV fluence. A decreased synthesis of poly(A)+ RNA and a severe inhibition of rRNA synthesis were observed on UV-irradiated and germinated spores, but no significant inhibition of 4-5 S RNA synthesis was detected. Actinomycin D suppressed almost completely the rRNA synthesis of emerged amoebae but the drug apparently did not affect the emergence of amoebae at any stage of germination. It was postulated that the delay of amoeba-emergence in UV-irradiated spore must be mainly due to the shift of the stage of maximum synthesis of poly(A)+ RNA to the later stage of germination.

Cell differentiation in a temperature-sensitive stalkless mutant of Dictyostelium discoideum

A temperature-sensitive aggregateless and stalkless mutant was isolated from Dictyostelium discoideum NC-4. The mutant cells cannot aggregate at 27 degrees C, but aggregate and form normal fruiting bodies at 21 degrees C. When the temperature was shifted to 27 degrees C after aggregation at 21 degrees C, almost all of the cells in the aggregate differentiated into spores. Neither stalk cells nor stalk tubes formed at 27 degrees C. Inhibition of stalk formation was not lifted by addition of cyclic AMP. Nevertheless, the proportion of prespore to total cells within the mutant slugs was normal, at both 21 degrees C and 27 degrees C. At 27 degrees C, a slug was transformed into a spherical cell mass at the end of migration, within which pre-existing prespore cells differentiated into spores. The remaining prestalk cells were then converted to prespore cells which later became spores. As the cell-type conversion continued, formation of a spore mass resulted. The development of the mutant is thus consistent with the idea that the presumptive cell differentiation is directly related to the terminal cell differentiation. During migration at 27 degrees C, the number of prestalk cells decreased in the anterior part of the slug but instead increased at the foot or the rear part, whereas the prestalk-prespore pattern remained normal at 21 degrees C. The fact that a normal proportion of prespore cells was maintained in spite of their deranged distribution at 27 degrees C indicates that the regulation of proportion is independent of the formation of pattern.

Effects of cyclic AMP on contact formation and differentiation in Dictyostelium discoideum.

It has been shown previously that Dictyostelium discoideum NC4 cells dissociated at the early aggregation stage form cell clumps and differentiate into prespore cells in a shaking culture containing glucose, albumin, EDTA and cyclic AMP. In this culture system, we found that the cells neither differentiate nor form cell clumps in the absence of cyclic AMP. Wheat-germ agglutinin (WGA) completely inhibited the cyclic AMP-induced formation of cell contact and the inhibition was nullified by the addition of N-acetyl-D-glucosamine. When the cells were prevented from forming contact by either rapid shaking or the addition of WGA, they were unable to differentiate even in the presence of cyclic AMP, indicating that contact formation is a prerequisite for prespore differentiation. Cells dissociated from migrating slugs formed cell clumps in shaking culture, with or without cyclic AMP, and the cell contact was sensitive to WGA. In the absence of cyclic AMP, prespore cells lost their differentiated state, even though the cells were in contact. This indicates that cyclic AMP has a second effect, that of pomoting differentiation, in addition to the effect of inducing contact formation. Both effects were required for prespore differentiation of strain NC4 cells.

Cell-surface discoidin in aggregating cells of Dictyostelium discoideum.

Both discoidin I and discoidin II have been detected on the surface of aggregating (10 h developmental stage) cells of Dictyostelium discoideum NC4 by radioiodination of the cell-surface followed by immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis. Approx. 92% of cell-surface discoidin I and 72% of cell-surface discoidin II can be eluted with 0.5 M-galactose, showing that most of each endogenous lectin is not present as integral membrane protein but rather is bound to cell-surface discoidin receptors. Two-dimensional polyacrylamide-gel-electrophoretic analysis of discoidin I suggests that the native tetramer may be a hetero-multimer composed of both Ia and Ib subunits. Cell-surface discoidin I also contains both types of subunit, but it is not clear whether both subunits have corresponding cell-surface receptors.

A Mutant of Dictyostelium discoideum with Alternative Pathways of Differentiation Depending on Culture Conditions

SUMMARY: A mutant (dev-1515), which forms either spores or stalk cells depending on the cultural conditions, was isolated from dev-1510, a mutant of Dictyostelium discoideum NC-4. When grown with Escherichia coli on a nutrient-rich medium, dev-515 formed a hemispherical cell mass in which most cells differentiated into spores. Culture with E. coli on a nutrient-intermediate medium inhibited amoebae from aggregating normally and they differentiated into stalk cells only. Development of washed vegetative cells of the mutant proceeded further without bacteria on either medium. Cells cultured in a roller tube were profoundly affected by the ionic strength of the medium. Almost all the cells in an agglutinate differentiated into stalk cells in a medium of low ionic strength, while most cells differentiated into spores in a medium of high ionic strength.

Kinetics of net RNA degradation during development in Dictyostelium discoideum.

Based on dry weight, first-order kinetics adequately describe the net decrease of RNA in Dictyostelium discoideum NC-4 until culmination, when a cessation of RNA degradation occurs. Between aggregation and sorocarp formation, the cells lose 40% of their RNA, or 19 mM-glucose equivalents expressed in terms of cell volume at aggregation. In sorocarps the spores contain a disproportionately large amount of RNA. Net RNA contents in sorocarps and in spores are unaffected by the availability of exogenous glucose. The relationship of dry weight, cell number and packed cell volume during differentiation is given.

Mitogenic and adjuvant activities of polysaccharides from the cellular slime mold, Dictyostelium discoideum NC-4

Cell surface and extracellular polysaccharide fractions obtained from Dictyostelium discoideum NC-4 cultured in bacteria-free medium showed strong B-cell mitogenic activities. Upon periodate treatment of the extracellular polysaccharide fraction this activity completely disappeared. The extracellular polysaccharide fraction could also enhance the antibody response in vitro against sheep red blood cells.

Extracellular agglutination factor of myxamoebae produced by Dictyostelium discoideum NC-4

A non-dialyzable specific agglutination factor of myxamoebae obtained from culture broth during the growth phase of Dictyostelium discoideum NC-4 was thermostable but the agglutination activity disappeared below pH 5.0. In the case of formalinized myxamoebae, digestion of the factor with Pronase decreased the activity, but periodate treatment of the factor did not affect the activity. Myxamoebal agglutination by this factor was inhibited by the addition of uronic acid, polyuronide (protuberic acid), and cell-surface polysaccharide prepared from the myxamoebae, but the agglutination was not affected by citric acid or glycine. The factor was purified by ethanol precipitation, column chromatography using DEAE-cellulose and Sepharose-2B, and zone electrophoresis. Chemical analysis of the purified factor gave 61.0% carbohydrate and 26.1% protein, and glucose, mannose, xylose and rhamnose (molar ratios of 9.3 : 3.2 : 2.1 : 1.0) were detected as the component sugars. The content of uronic acid was 12.9%. When the myxamoebae of the growth phase were starved in Millipore-supporting medium, the agglutination activity was detected in the supernatant of the medium.

Pacemaker mutants of Dictyostelium discoideum

In this paper, I report measurements of the time intervals between aggregation waves in two morphogenetic mutants of Dictyostelium discoideum NC-4. The mutants both have abnormal wave periodicity and their abnormalities are interpreted in terms of defects in the cellular properties which underlie wave propagation. The main results are as follows: 1. (1) The two mutants each have the same categories of waveforms as wild-type Dictyostelium discoideum NC-4 (concentric ring and spiral waves). The two kinds of waveforms have characteristic temporal intervals in the wild-type and mutant strains. 2. (2) In the mutant 91A, concentric ring and spiral waves resemble equivalent wild-type waves in their temporal periodicity, but the mutant has a high proportion of spiral waves (60% as compared to 16% in NC-4). This abnormality is shown to be due at least partly to a low rate of emergence of autonomous signaling competence in 91A cells during early aggregation. 3. (3) The mutant Fr17 has normal spiral waves but abnormal concentric ring waves. The concentric ring waves of Fr17 are aperiodic and separated by irregularly sequenced intervals between about 3 and 40 min long. Those of NC-4 are usually either approximately periodic, with 5-or 10-min periodicity or are separated by steadily decreasing intervals between and 7 and 2.5 min ( Durston, 1974). I suggest that Fr17 lacks two cell properties manifested in NC-4 (periodic and continuous autonomous signaling) and instead has a new property (aperiodic autonomous signaling).

Accumulation of uridine diphosphoglucose pyrophosphorylase in Dictyostelium discoideum via preferential synthesis

At the end of the exponential growth phase, the enzyme UDP-glueose pyrophosphorylase is present in the vegetative cells of Dictyostelium discoideum NC4 (haploid) at a low level (about 0.05% of total protein). During the initial stages of fruiting body construction, while the cells are entering into multicellular aggregates, the enzyme level remains constant, but increases dramatically thereafter reaching a peak (about 0.5% of total protein) at the end of fruiting body construction, and then partially decreasing. Previous studies have shown that both the accumulation and disappearance are keyed to the flow of morphogenetic events. In this study, cells were labeled with amino acids for different periods throughout the sequence. The enzyme was quantitatively immune-precipitated from crude cell extracts, the precipitate was washed and redissolved, and the enzyme protein separated by acrylamide gel electrophoresis in order to estimate the differential incorporation ratio, i.e. disints min in enzyme protein per 10 8 cells disints min in total protein per 10 8 cells × 100 for each labeling period. During the initial stages, when the enzyme level remained relatively constant, this ratio was about 0.03 to 0.04%. As the enzyme began to accumulate it rose progressively, attaining levels of 0.6 to 0.8% toward the end of fruiting body construction before declining. The data are not consistent with the theory of Gustafson and Wright (1973) that differential turnover controls the level of this enzyme during the development of D. discoideum. They are consistent with the conclusion that directed changes in the differential rate of synthesis of UDP-glucose pyrophosphorylase is the controlling element. The estimates of enzyme content are based on a value for the specific enzyme activity of 100,000 units/mg enzyme, which had been determined previously using samples of the enzyme purified to apparent physical homogeneity. This figure has been confirmed in the present study by quantitative immuneprecipitation of the enzyme from crude extracts of homogeneously labeled cells. The method can be generally used to determine if a specific biological activity estimate obtained with a purified protein is consistent with its activity when measured before or during purification.

Independence of propagation ability and developmental processes in irradiated cellular slime-moulds.

SummaryWhen assayed for reproductive ability, the cellular slime mould Dictyostelium discoideum NC-4 is resistant to 60Co gamma-rays (D10 = 300 krads) whereas a derived mutant, γs-13, is much more sensitive (D10 = 4 krads). If ability to proceed through development to form normal-appearing spores (a process requiring neither DNA synthesis nor cell division) is assayed after irradiation of amoeboid cells, the γs-13 strain is more sensitive than NC-4, but the differential sensitivity is only about 4× at the 20 per cent level (270 krads for NC-4). When the reproductive ability of these spores is determined, those of γs-13 are mostly inviable at doses above 20 krads, while those of NC-4 are largely viable up to 200 krads. Quantitatively, the proportion of viable spores among those produced closely follows the proportion of viable cells in the irradiated population used to initiate development. The greatest diminution of spore production occurs for irradiation done at the beginning of aggregation; however, the...

Gamma-Ray-Resistant and -Sensitive Strains of Slime Mold (Dictyostelium discoideum)

The vegetative cells and spores of the cellular slime mold Dictyostelium discoideum NC-4 are very resistant to60 Co gamma rays, with a 10% survival dose ( D10) of 300 krad in air. Using ultraviolet light or nitrosoguanidine as a mutagenic agent and replica plating with gamma irradiation as a selective agent, we have isolated several stable daughter strains that are more sensitive to gamma rays. A comparison of some of the radiation responses of the parent (NC-4; wild type), γs-18 ( ${\rm D}_{10}=75 {\rm krad}$), and γs-13 ( ${\rm D}_{10}=4 {\rm krad}$) strains has been made. All three strains show the same gamma-ray-induced division delay of about 1 cell generation time for 54 krad. Strains wt (wild type) and γs-18 show split-dose recovery of colony-forming ability but γs-13 does not. For wt, this recovery is temperature-dependent with a maximum at 23°C, the most favorable growth temperature. Some recovery still occurs at 5-10°C, although growth does not. Strains wt and γs-18 are more sensitive when irr...