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.

Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum.

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.

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.

Dictyostelium discoideum mRNAs developmentally regulated during spore germination have short half-lives.

mRNA decay was studied during spore germination in Dictyoselium discoideum by the use of three previously isolated cDNA clones, pLK109, pLK229, and pRK270, which are specific for mRNAs developmentally regulated during spore germination. The half-life of a constitutive mRNA, pLK125, which is present throughout germination, growth, and development, as also determined. Nogalamycin, a DNA-intercalating compound, was used to inhibit RNA synthesis. Total RNA was isolated at intervals after addition of the drug, and the decay of mRNAs specific for the cDNA clones was determined by both Northern blot and RNA dot hybridization. If nogalamycin was added immediately after activation of dormant spores, neither pLK229 nor pLK109 mRNA decayed, but pLK125 mRNA did decay. Although pLK109 mRNA did not decay under these conditions, the RNA was smaller 1 h after activation than in dormant spores, indicating that it was processed normally. At 1 h after activation, pLK229-, pLK125-specific mRNAs decayed exponentially, with half-lives of 24, 39, and 165 min, respectively. Under the same conditions, decay of pLK109-specific mRNA was biphasic. Thirty-eight percent of the mRNA decayed with a half-life of 5.5 min, and the remainder decayed with a half-life of 115 min. It seems likely that nogalamycin inhibits the synthesis of an unstable component of the mRNA degradative pathway which is needed continuously for the decay of pLK109 mRNA. By extrapolating the curve representing the rapidly decaying component, a half-life of 18 min was calculated for pLK109-specific mRNA. The mRNAs developmentally regulated during spore germination have half-lives shorter than that of the constitutive messenger and shorter than the average half-life of 3 to 4 h previously determined for total Dicyostelium polyadenylated mRNA.

Conservation and variation of ribosomal proteins in several species of the cellular slime molds Dictyostelium and Polysphondylium.

Genus- and species-specific composition of ribosomal proteins was investigated in four species of the genus Dictyostelium (D. discoideum, D. purpureum, D. murcoroides and D. giganteum) and two species of the genus Polysphondylium (P. pallidum and P. violaceum). Ribosomal proteins were resolved by a high-resolution, two-dimensional gel method. In general, the numbers and distributions for the majority of ribosomal proteins were similar within the species of each genus, although some differences were detected. More differences were observed between Dictyostelium and Polysphondylium than among the individual species within each genus. Stage-specific ribosomal proteins previously demonstrated in D. discoideum were found to be developmentally regulated in other Dictyostelium species, and in both Polysphondylium species. The study shows that ribosomal proteins may be a potentially useful new biochemical parameter for the molecular taxonomy of the cellular slime molds.

Developmental regulation of protein synthesis during Polysphondylium pallidum cyst germination: a two-dimensional gel electrophoresis study.

Microcyst germination in Polysphondylium pallidum can be used as a model for studying gene expression because temporally regulated modulations in protein synthesis occur in this developmental pathway. Germinating cysts were labeled with [35S]methionine for half-hourly periods during the synchronous germination sequence, and the proteins labeled in each period were resolved by two-dimensional polyacrylamide gel electrophoresis. Three major classes of proteins observed were distinguished by the time of onset and duration of their synthesis: (a) proteins made throughout germination; (b) proteins synthesized only during a portion of the germination pathway; and (c) polypeptides whose synthesis started at 1 or 1.5 h and then continued throughout germination.

A genetic interaction between a ubiquitin-like protein and ubiquitin-mediated proteolysis in Dictyostelium discoideum(1).

A ubiquitination factor, NosA, is essential for cellular differentiation in Dictyostelium discoideum. In the absence of nosA, development is blocked, resulting in a developmental arrest at the tight-aggregate stage, when cells differentiate into two precursor cell types, prespore and prestalk cells. Development is restored when a second gene, encoding the ubiquitin-like protein SonA, is inactivated in nosA-mutant cells. SonA has homology over its entire length to Dsk2 from Saccharomyces cerevisiae, a ubiquitin-like protein that is involved in the assembly of the spindle pole body. Dsk2 and SonA are both stable proteins that do not seem to be subjected to degradation via the ubiquitin pathway. SonA does not become ubiquitinated and the intracellular levels of SonA are not affected by the absence of NosA. The high degree of suppression suggests that SonA rescues most or all of the defects caused by the absence of nosA. We propose that NosA and SonA act in concert to control the activity of a developmental regulator that must be deactivated for cells to cross a developmental boundary.

Isolation of a Dictyostelium discoideum 14-3-3 homologue.

A 1.0 kb cDNA clone (Dd14-3-3) encoding a 14-3-3 homologue was isolated from a Dictyostelium discoideum cDNA library. The putative Dd14-3-3 protein has highest sequence identity to a barley 14-3-3 isoform (74%). Southern blot analysis suggests that only one 14-3-3 gene is present in the Dictyostelium genome. Highest Dd14-3-3 expression is observed in vegetatively growing cells, and expression decreases during multicellular development. In contrast, Dd14-3-3 protein levels detected immunochemically remained constant during Dictyostelium development. Expression of the Dd14-3-3 cDNA in Saccharomyces cerevisiae complemented the lethal disruption of the two yeast genes encoding 14-3-3 proteins (BMH1 and BMH2). This shows that Dd14-3-3 can fulfil the same function(s) as the yeast 14-3-3 proteins.

Organization of a gene family developmentally regulated during Dictyostelium discoideum spore germination.

mRNA specific to cDNA clone pLK109 is present in Dictyostelium discoideum spores, increases about two- to threefold at 0.5 to 1 h during spore germination, and then rapidly decreases. The mRNA is not detectable in vegetative cells or in early multicellular development on filters, but is present late during development, approximately at the time of sporulation. 109 mRNA in spores is 700 nucleotides in length but this is processed during germination by shortening of the poly(A) tail to about 600 nucleotides at 1 to 1.5 hours. pLK109 is a member of a multigene family containing three separate genes, and we have isolated and sequenced all of them. All three sequences code for deduced proteins of 127 amino acid residues, with only a few amino acid differences among them. Gene 1 represents the "transcribed" gene, since all 33 cDNAs we isolated are identical with the cDNA pLK109 and the coding region of this gene. Other open reading frames are in close proximity to each of the 109 sequences. About 200 base-pairs 3' to the gene 1 109 sequence is an open reading frame in the opposite orientation. Gene 2 fragment contains a sequence that codes for a protein similar to trypanosome alpha-tubulin 728 base-pairs 5' to the 109 sequence. Gene 3 fragment possesses two additional putative coding regions, one 5' and another 3' to the 109 gene. There is a remarkable similarity between the 5' upstream regions of all three genes. Each possesses a normal Dictyostelium TATA box and the usual T stretch. In addition, there are many other portions of about 400 to 500 base-pairs of the 5' regions that are either identical for long stretches or very similar.

Bacterial resistance to the synergistic antibiotics of the PA 114, streptogramin, and vernamycin complexes.

A mutant of Bacillus subtilis was isolated that was resistant to the growth inhibitory activity of the synergistic antibiotics of the PA 114, streptogramin, and vernamycin complexes. Escherichia coli is naturally resistant to the action of these antibiotics. In both cases, it was shown that resistance was due to an inability of the bacteria to transport the antibiotics into the cell.

Nogalamycin inhibits ribonucleic acid synthesis in growing and developing cells of the slime mold Dictyostelium discoideum.

Nogalamycin, an anthracycline antibiotic that intercalates into deoxyribonucleic acid, is a potent inhibitor of ribonucleic acid (RNA) synthesis in the slime mold Dictyostelium discoideum. The antibiotic inhibits RNA synthesis in growing cells and in inactivated spores, and in this way inhibits spore germination. Protein synthesis is much less inhibited. Nogalamycin inhibits ribosomal RNA, transfer RNA, and messenger RNA equally. Polysomes break down in the presence of the drug with a half-life of 220 min, and messenger RNA decays with a half-life of 290 min. The data show that nogalamycin can be employed to inhibit messenger RNA synthesis and is useful in determining messenger RNA decay rates in the slime mold.

Mutants of Escherichia coli sensitive to antibiotics.

Mutants of Escherichia coli sensitive to the antibiotic synergistin A, an inhibitor of protein synthesis, were isolated. These mutants were pleiotropic, being also sensitive to a large number of unrelated antibiotics and to lysis by detergents. These pleiotropic responses indicated that the mutations affected cell wall or membrane synthesis. Consequently, selection for antibiotic-sensitive mutants constitutes a useful means for isolating cell wall or membrane mutants.

Polysome metabolism in Escherichia coli: effect of antibiotics on polysome stability.

A number of antibiotics, most of which specifically inhibit protein synthesis by interacting with the 50S ribosome subunit, were used to study polysome metabolism in vivo. The antibiotics affected polysomes in two ways: (i) by allowing extensive breakdown of polysomes, or (ii) by stabilizing polysomes, even in the presence of the antibiotics of the first group. The results indicate that the effect of these antibiotics on polysome stability in vivo can, in many cases, be correlated to their in vitro mode of action.

Polysome metabolism in Escherichia coli: amicetin, an antibiotic that stabilizes polysomes.

Amicetin, an aminohexosepyrimidine nucleoside antibiotic, inhibited protein synthesis in intact bacterial cells and in cell-free extracts derived from these cells. At concentrations of antibiotic which rapidly and completely inhibited protein synthesis, the polysomes in the cell were stabilized. Amicetin also inhibited the breakdown of polysomes induced by the streptogramin A antibiotics, rifampin, or puromycin, antibiotics that require peptide bond formation for expression of their action on polysomes. Thus, amicetin is another addition to the list of drugs which may be useful in studying polysome and messenger ribonucleic acid metabolism in vivo. An in vitro system is described which may be generally useful in studying polysome metabolism when antibiotic-sensitive strains are not available.