Herbert L. Ennis

Dictyostelium amoebae lacking an F-box protein form spores rather than stalk in chimeras with wild type

Using a selection for Dictyostelium mutants that preferentially form spores, we have recovered a mutant called CheaterA. In chimeras with isogenic wild-type cells, the CheaterA mutant preferentially forms viable spores rather than inviable stalk cells. The mutant causes wild-type cells that have begun to express spore-specific genes to accumulate in the prestalk compartment of the developing organism. In the wild-type cells, the chtA transcript is absent in growing cells and appears early in development. No transcript was detected in the mutant by Northern blot. The chtA gene codes for a protein with an F-box and WD40 domains. This class of protein usually forms part of an Skp1, cullin, F-box (SCF) complex that targets specific protein substrates for ubiquitination and degradation.

Nucleotide sequences of Dictyostelium discoideum developmentally regulated cDNAs rich in (AAC) imply proteins that contain clusters of asparagine, glutamine, or threonine

SummaryA Dictyostelium discoideum repetitive element composed of long repeats of the codon (AAC) is found in developmentally regulated transcripts. The concentration of (AAC) sequences is low in mRNA from dormant spores and growing cells and increases markedly during spore germination and multicellular development. The sequence hybridizes to many different sized Dictyostelium DNA restriction fragments indicating that it is scattered throughout the genome. Four cDNA clones isolated contain (AAC) sequences in the deduced coding region. Interestingly, the (AAC)-rich sequences are present in all three reading frames in the deduced proteins, i.e., AAC (asparagine), ACA (threonine) and CAA (glutamine). Three of the clones contain only one of these in-frame so that the individual proteins carry either asparagine, threonine, or glutamine clusters, not mixtures. However, one clone is both glutamine- and asparagine-rich. The (AAC) portion of the transcripts are reiterated 300 times in the haploid genome while the other portions of the cDNAs represent single copy genes, whose sequences show no similarity other than the (AAC) repeats. The repeated sequence is similar to the opa or M sequence found in Drosophila melanogaster notch and homeo box genes and in fly developmentally regulated transcripts. The transcripts are present on polysomes suggesting that they are translated. Although the function of these repeats is unknown, long amino acid repeats are a characteristic feature of extracellular proteins of lower eukaryotes.

Developmental changes in RNA and protein synthesis during germination of Dictyostelium discoideum spores

Abstract Spore germination in Dictyostelium discoideum is a synchronous developmental process involving three distinct stages: activation, swelling, and emergence of amoebae. The regulation of protein and RNA synthesis during normal germination in the wild-type D. discoideum has been compared with the events that occur during abortive germination of mutant HE-1 and cycloheximide-treated wild-type spores, both of which are blocked at the emergence stage. Some aspects of the regulation of the synthesis of different types of RNA were investigated. RNA was isolated from germinating spores labeled at hourly intervals after activation. The RNA was fractionated by oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation. Analysis of the RNA shows that the synthesis of mRNA, tRNA, and rRNA is developmentally regulated during germination. During the initial stages, the majority of the RNA made is mRNA and tRNA, while during later stages of germination, more rRNA is synthesized. In order to detect the presence of functional mRNA at different stages, RNA was isolated from dormant and germinating spores. Translatable mRNA was assayed by the ability of the RNA to stimulate incorporation of radioactive amino acids into protein in a wheat germ cell-free extract. The results show that dormant spores contain translatable polyadenylated mRNA, and the fraction of total RNA with mRNA activity increases during germination. From an analysis of the proteins synthesized during germination, it is evident that the synthesis of proteins is also developmentally regulated. Four classes of proteins could be distinguished on gels, depending on the time of onset and duration of their synthesis. A comparison of proteins made in vivo with those synthesized in vitro indicates that there are qualitative changes in the population of translatable mRNAs during germination, presumably due to differential gene transcription. Some of these changes observed in vitro correlate well with the differences in proteins synthesized in intact germinating spores. RNA and protein synthesis were compared in mutant HE-1 and in wild-type germination. In the mutant, unlike the wild type, there is no change in the proportion of mRNA made during germination, and rRNA represents a smaller portion of the total RNA made at any interval than in the wild type. In addition, it is noteworthy that the pattern of protein synthesis during abortive germination in the mutant is very different from that observed in the wild type. The control over the synthesis of different types of RNA and proteins during germination appears abnormal in the mutant, suggesting that developmental regulation is defective in the mutant.

Protein and RNA synthesis during spore germination in the cellular slime mold Dictyostelium discoideum

Summary Germination of Dictyostelium discoideum is a synchronous developmental process, known to involve three distinct stages: activation, swelling and emergence of amoebae. This entire sequence is completed within 3.5 h after activation of dormant spores. Both protein and RNA synthesis appear to be required for germination. Protein synthesis, as judged from the incorporation of labeled precursors, begins during swelling. Spores do not contain polysomes, but polysomes appear during the swelling stage, coincident with the beginning of incorporation of amino acid into protein. SDS-polyacrylamide gel electrophoresis of labeled extracts shows that stage-specific changes occur in the pattern of proteins synthesized during germination. RNA synthesis also appears to start during swelling and inhibitors of RNA synthesis are shown to block germination.

Separation and purification of the mRNAs for vesciular stomatitis virus NS and M proteins

Abstract All five vesicular stomatitis virus mRNAs were separated and purified by acid-urea agarose gel electrophoresis. The identities of the NS and M mRNAs were established by their in vitro translation.

Membrane protein synthesis after infection of Escherichia coli B with phage T4: The rIIB protein

Abstract Inner and outer cell membrane proteins synthesized after infection of Escherichia coli B with wild-type phage T4, r IIA and B mutants and a spackle mutant were analyzed by SDS-polyacrylamide gel electrophoresis. At least three new proteins of approximate molecular weights 22,000, 29,000, and 37,000 daltons are made after infection with wild-type phage. The protein of molecular weight 37,000 is not present in membranes of cells infected with an r IIB deletion mutant. This protein is the major protein made from 5 to 15 minutes after infection with the wild type. In addition, the major host protein of the outer envelope is not made during wild-type phage infection.

Vernamycin A inhibits the non-enzymatic binding of fMet-tRNA to ribosomes

Abstract The ribosomal site of action of the antibiotic vernamycin A was studied by investigating the effect of the drug on the non-enzymatic binding of fMet-tRNA. 1. 1. Vernamycin A inhibits the non-enzymatic binding of fMet-tRNA to 70-S ribosomes and to 50-S subunits, and can displace previously bound fMet-tRNA from 70-S ribosomes. 2. 2. Vernamycin A inhibits the binding of fMet-tRNA to a puromycin reactive site (P site), which is not affected by tetracycline. 3. 3. Although vernamycin A inhibits the puromycin reaction in model systems it does not inhibit peptide bond formation occurring on polysomes. Consequently, the antibiotic probably inhibits peptide bond formation in model in vitro systems not by directly inhibiting peptide bond formation, but by inhibiting the binding of the tRNA to the ribosome. 4. 4. The data indicate that the ribosomal binding sites for vernamycin A on the one hand and carbomycin B, chloramphenicol, erythromycin, leucomycin A 3 , spiramycin III and tylosin on the other hand, are different.

Decay of vesicular stomatitis virus mRNAs in Vivo

Abstract The stability of vesicular stomatitis virus (VSV) messenger RNA during infection of Chinese hamster ovary (CHO) cells was investigated using the temperature-sensitive mutant, tsG114. By incubating infected cells at the nonpermissive temperature (39°), RNA synthesis was blocked and VSV mRNA decayed rapidly, as determined by sucrose density gradient analysis. Each of the five VSV mRNAs was also found to decay functionally at approximately the same rate suggesting that the differential rates of accumulation of VSV mRNAs observed in vivo are due to differential rates of transcription of specific VSV genes.

Binding of the antibiotic vernamycin in Bα to Escherichia coli ribosomes

Abstract The interaction of the antibiotic vernamycin Bα with Escherichia coli ribosomes has been studied. The antibiotic is bound to 70S ribosomes and 50S subunits but not to the 30S subunit or to polysomes. The binding of the antibiotic requires K+ or NH+4 and Mg2+. At saturation approximately 0.5 mole of antibiotic is bound per mole of ribosomes. The vernamycin Bα-ribosome complex is unstable. The bound antibiotic is readily displaced by nonradioactive vernamycin Bα and by a number of other antibiotics which are known to interact with the 50S subunit. The dissociation of the vernamycin Bα-ribosome complex is prevented by the simultaneous binding of vernamycin A. The binding sites for A and Bα are distinguishable since both drugs are able to bind simultaneously and neither prevents binding of the other, Ribosomes isolated from an erythromycin-resistant mutant are incapable of binding vernamycin A and Bα, indicating that the mutated protein responsible for resistance to erythromycin distorts the ribosome making it also unreceptive for the vernamycins.

The Dictyostelium discoideum spore germination-specific cellulase is organized into functional domains.

During Dictyostelium discoideum spore germination, degradation of the cellulose-containing spore wall is required to allow the amoeba to emerge. The CelA gene, which is transcribed and expressed exclusively during spore germination, codes for a 705-amino-acid protein that has cellulase activity [endo-(1,4)-beta-D-glucanase]. Amoebae transformed by a vector containing the CelA coding sequence or portions of it transcribed from a heterologous promoter expressed and secreted full-length or suitably truncated proteins during vegetative growth when, under normal conditions, these proteins are not made. The gene constructs divided the CelA protein into three domains: a 461-amino-acid N-terminal region that has significant similarity to those of other cellulases and that has been shown to be the catalytic domain; a contiguous 91-residue repeat containing the motif threonine-glutamic acid-threonine-proline, which is glycosylated; and, joined to the repeat, a C-terminal 153-amino-acid sequence that most probably defines a cellulose-binding domain.