Vagn Leick

Free ribosomal DNA molecules from Tetrahymena pyriformis GL are giant palindromes.

Restriction ondonuclease EcoRI was used to study the structure of the free ribosomal DNA molecules from Tetrahymena pyriformis, strain GL. From the following observations we conclude that the free rDNA molecules from Tetrahymena are giant palindromes‡, each containing two genes for preribosomal RNA arranged in rotational symmetry as inverted repeating sequences. Analyses of the sizes of products of partial or complete digestion and quantitative analyses of the products of complete digestion of uniformly 32P-labeled rDNA yielded an RI endonucleolytic cleavage map which showed that the EcoRI recognition sites are arranged symmetrically about the center of the rDNA molecule. When heat-denatured rDNA was rapidly cooled under conditions in which no renaturation would occur between separated complementary strands of DNA, molecules of half the size of the original rDNA molecule were produced. These were double-stranded DNA molecules as evidenced by their resistance to digestion with S1 nuclease. Moreover, they could be digested with EcoRI to produce fragments of sizes which would be predicted from the assumption that each single strand of the original rDNA molecule had folded back on itself to form a “hair-pin” double-stranded DNA structure. Hybridization experiments between ribosomal RNA and purified rDNA showed that each rDNA molecule contains two genes for rDNA. Hybridization of the isolated EcoRI fragments of rDNA with 25 S or 17 S rRNA suggested that the two structural genes for 17 S rRNA are located near the center of the rDNA molecule and the two genes for 25 S rRNA are found in distal positions.

Rapid equilibrium isopycnic CsCl gradients

Abstract The use of equilibrium isopycnic CsCl gradients with an initial preformed step in CsCl concentration dramatically reduces the time required to achieve equilibrium conditions. The initial step in CsCl concentration increases the net force on the macrospecies and thus allows the formation of a narrow band at approximately the equilibrium position during the early phase of centrifugation. This type of gradient has been demonstrated to be useful with macrospecies including DNA, RNA, and formaldehyde-fixed ribonucleoprotein particles.

Signaling in Unicellular Eukaryotes

Aspects of intercellular and intracellular signaling systems in cell survival, proliferation, differentiation, chemosensory behavior, and programmed cell death in free-living unicellular eukaryotes have been reviewed. Comparisons have been made with both bacteria and metazoa. The central organisms were flagellates (Trypanosoma, Leishmania, and Crithidia), slime molds (Dictyostelium), yeast cells (Saccharomyces cerevisiae), and ciliates (Paramecium, Euplotes, and Tetrahymena). There are two novel aspects in this review. First, cellular responses are viewed in an evolutionary perspective, rather than from the more prevailing one, in which the unicellular eukaryotes are seen by the mammalian organisms. Second, results obtained with cell cultures in minimal, chemically defined nutrient media at low cell densities where intercellular signaling is strongly reduced are discussed. These results shed light on control mechanisms and their cooperation inside the living cell. Intracellular systems have many common features in unicellular and multicellular organisms.

Autonomous rDNA molecules containing single copies of the ribosomal RNA genes in the macronucleus of Tetrahymena pyriformis

Abstract The DNA containing the genes for rRNA (commonly called rDNA) of Tetrahymena sediments in sucrose density gradients considerably slower than the main part of the DNA when DNA from gently lysed whole cells or isolated nuclei are fractionated by this method. In rDNA purified by CsCl gradient centrifugation about 20% of the DNA (40% of the bases in one strand) consists of sequences homologous to 25S and 17S rRNA as determined by DNA-RNA hybridization. The purified rDNA co-sediments in sucrose gradients with O29 phage DNA (M.W. = 11 × 106). Examination by electron microscopy of the rDNA demonstrates that the molecules are linear with a length of 5.65 ±0.6 μm corresponding to a molecular weight of 11 × 106.

Formation of ribosomes in Tetrahymena pyriformis

Abstract The formation of ribosomal sub-units—derived from ribosomes by dialysis against low Mg 2+ concentration—has been studied by incorporation of RNA precursors into exponentially growing Tetrahymena cells. The 50-S derived sub-units show a delayed kinetics of labeling as compared to the 30-S derived subunits. The RNA constituents of 50-S derived sub-units are 25-S RNA and 5-S RNA, and the RNA constituent of derived 30-S sub-units is 17-S RNA as determined by methylated albumin-kieselguhr column chromatography. The delay in labeling of the 50-S derived sub-unit is a result of a slower rate at some stage later than the transcription of 25-S RNA presumably involving delayed completion as well as delayed transport of large sub-units. A 60-S, rapidly labeled cytoplasmic ribonucleoprotein particle is found which shows characteristics of a precursor to 50-S derived sub-units. In cells with induced synchrony of cell division, an increase in ribosome formation is observed shortly after the synchronized cell division.

A quantitative assay for ciliate chemotaxis

A quantitative bioassay for ciliate chemotaxis based on the capillary principle is described using Tetrahymena thermophila as test organism. The attractant-containing assay tube designed for the bioassay attracts up to 4 X 10(4) cells in 2 h which makes electronic cell counting of the chemotactic response feasible. The attractants used are solutions of proteose peptone and yeast extract which also are growth media for this organism.

CHEMOATTRACTION IN TETRAHYMENA: ON THE ROLE OF CHEMOKINESIS

Chemoattraction of Tetrahymena pyriformis, strain GL, was measured during starvation and under different growth conditions. Log phase cells starved in buffer are attracted by certain amino acids, peptides, and proteins. Cysteine, methionine, and phenylalanine are attractants at i0@ M. The peptides in proteose peptone (PP) and yeast extract (YE) are active at 10_6M. Epidermal growth factor (EGF) is active at >3 X i0@ M. Among the proteins, platelet derived growth factor (PDGF) is the most active (3 X 108 M). Cells growing in defined medium are attracted by PP, YE, and some proteins (PDGF). Swimming speed was measured for starved cells with and without added attractants or repellents. With addition of PP the swimming speed increases from 0.42 to 0.51 mm/s., but for PDGF it is unchanged. The swimming speed of starved cells increases when the cells approach a solidified attractant (PP) as measured by the speed at a given distance. The speed of cells moving towards the attractant is higher than that of cells moving away from it. In conclusion, certain amino acids, peptides, and proteins are chemoattractants for Tetrahymena. Chemokinesis likely plays a considerable role in the case of PP (and YE), since they increase swimming speeds, whereas attraction by PDGF may involve chemotaxis.

Protein phosphatase 2A (PP2A) regulates interleukin-4-mediated STAT6 signaling.

Interleukin-4 (IL-4) plays a pivotal role in the induction and maintenance of allergy by promoting Th2 differentiation and B cell isotype switching to IgE. Studies on STAT6-deficient mice have demonstrated the essential role of STAT6 in mediating the biological functions of IL-4. IL-4 induces tyrosine phosphorylation of STAT6, which in turn leads to transcription of IL-4-specific genes. In addition, serine phosphorylation of STAT6 has recently been reported. Here we study the functional role of STAT6 serine phosphorylation and the kinases and phosphatases involved. We show that inhibition of protein phosphatase 2A (PP2A) induces serine phosphorylation of STAT6 and severely inhibits DNA binding of STAT6. In contrast, IL-4-induced tyrosine phosphorylation of Janus kinase-1 and STAT6 is not affected, suggesting that PP2A acts downstream of Janus kinases in IL-4 signaling. In conclusion, we provide the first evidence that PP2A plays a crucial role in the regulation of STAT6 function.

Platelet-derived growth factor stimulates chemotaxis and nucleic acid synthesis in the protozoan Tetrahymena

Platelet-derived growth factor (PDGF) is in concentrations of a few nanograms per ml a very active chemoattractant for the free-living ciliated protozoan Tetrahymena; at the same time it induces a rapid increase in incorporation of radioactive nucleic acid precursors into RNA and DNA. We find it remarkable that this lower eukaryote responds to platelet-derived growth factor in very much the same way as fibroblastic cells.

Cilia-Mediated Oriented Chemokinesis in Tetrahymena thermophila

The role of the cilia in the locomotion (“gliding”) of Tetrahymena thermophila in a semi‐solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer‐aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi‐solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination (“stop”) of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de‐adaptation, which occurs during the “stop”. In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant‐induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short‐term memory during adaptation. This is important for the efficiency of migration towards an attractant.