Identification of single nucleotide mutations that prevent developmentally programmed DNA elimination in Paramecium tetraurelia

The excision of internal eliminated sequences (IESs) occurs during the differentiation of a new somatic macronuclear genome in ciliated protozoa. In Paramecium tetraurelia, IESs show few conserved features with the exception of an invariant 5'-TA-3' dinucleotide that is part of an 8-bp inverted terminal repeat consensus sequence with similarity to the ends of mariner/Tc1 transposons. We have isolated and analyzed two mutant cell lines that are defective in excision of individual IESs in the A-51 surface antigen gene. Each cell line contains a mutation in the flanking 5'-TA-3' dinucleotide of IES6435 and IES1835 creating a 5'-CA-3' flanking sequence that prevents excision. The results demonstrate that the first position of the 5'-TA-3' is required IES excision just as previous mutants have shown that the second position (the A residue) is required. Combining these results with other Paramecium IES mutants suggests that there are few positions essential for IES excision in Paramecium. Analysis of many IESs reveals that there is a strong bias against particular nucleotides at some positions near the IES termini. Some of these strongly biased positions correspond to known IES mutations, others correlate with unusual features of excision.     

Purification and characterization of the myoglobins of Paramecium tetraurelia.

1. The molecular variants of the myoglobins of Paramecium tetraurelia have been purified as five separate components and designated Mb 1 to Mb 5. 2. The five molecular forms are homogeneous on polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IF). 3. The myoglobin species appear to have identical molecular weights and amino acid compositions and differ only in their isoelectric points and relative concentrations in vivo. 4. The myoglobin species have an apparent molecular weight of 15,000 +/- 500 and possess a single heme group per mole which appears to be protoporphyrin IX. 5. The amino acid composition of the five species is: lys12, his3, arg4, asp17, thr9, ser6, glu16, pro4, gly11, ala15, cys0, val8, met2, ile7, leu10, tyr5, phe7. 6. The spectra of several ferrous and ferric derivatives of the mixture Mb 1-Mb 5 are presented.     

Calcium Regulation in the Protozoan Model,Paramecium tetraurelia

Early in eukaryotic evolution, the cell has evolved a considerable inventory of proteins engaged in the regulation of intracellular Ca²⁺ concentrations, not only to avoid toxic effects but beyond that to exploit the signaling capacity of Ca²⁺ by small changes in local concentration. Among protozoa, the ciliate Paramecium may now be one of the best analyzed models. Ciliary activity and exo‐/endocytosis are governed by Ca²⁺, the latter by Ca²⁺ mobilization from alveolar sacs and a superimposed store‐operated Ca²⁺‐influx. Paramecium cells possess plasma membrane‐ and endoplasmic reticulum‐resident Ca²⁺‐ATPases/pumps (PMCA, SERCA), a variety of Ca²⁺ influx channels, including mechanosensitive and voltage‐dependent channels in the plasma membrane, furthermore a plethora of Ca²⁺‐release channels (CRC) of the inositol 1,4,5‐trisphosphate and ryanodine receptor type in different compartments, notably the contractile vacuole complex and the alveolar sacs, as well as in vesicles participating in vesicular trafficking. Additional types of CRC probably also occur but they have not been identified at a molecular level as yet, as is the equivalent of synaptotagmin as a Ca²⁺ sensor for exocytosis. Among established targets and sensors of Ca²⁺ in Paramecium are calmodulin, calcineurin, as well as Ca²⁺/calmodulin‐dependent protein kinases, all with multiple functions. Thus, basic elements of Ca²⁺ signaling are available for Paramecium.     

Expression of the green fluorescent protein in Paramecium tetraurelia

In this paper we describe the expression of green fluorescent protein (GFP) as a reporter in vivo to monitor transformation in Paramecium cells. This is not trivial because of the limited number of strong promoters available for heterologous expression and the very high AT content of the genomic DNA, the consequence of which is a very aberrant codon usage. Taking into account differences in codon usage we selected and modified the original GFP open reading frame (ORF) from Aequorea victoria and placed the altered ORF into the Paramecium expression vector pPXV. Injection of the linearized plasmid into the macronucleus resulted in a cytoplasmic fluorescence signal in the clonal descendants, which was proportional to the number of copies injected. Southern hybridization indicated the establishment and replication of the plasmid during vegetative growth. Expression was also monitored by Northern and Western analysis. The results indicate that the modified GFP can be used in Paramecium as a reporter for transformation as an alternative to selection with antibiotics and that it may also be used to construct and localize fusion proteins.     

Effects of mutagens on the clonal lifespan of Paramecium tetraurelia.

There has been interest in the phenomenon that a cell cannot undergo unlimited reproduction under adequate conditions and undergoes senescence. In holotrichous ciliates, Paramecium has a limit of vegetative reproduction without sexual reproduction but Tetrahymena does not always have a limited lifespan. Comparing the two species would increase our knowledge of the mechanism of cellular clonal aging. We previously showed that mutations induced by X-rays shorten clonal lifespan. In this study, we examined whether mutagens shorten the clonal lifespan of Paramecium tetraurelia. P. tetraurelia was exposed to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 0.045 mg/ml, for 30 min. The animal was exposed to MNNG 6 times in total while young (under 80 divisions from the start of a clonal life cycle) or 4 times during the senescent stage. MNNG shortened the clonal lifespan as expressed by the decrease in fission number from 186 +/- 55 (4 cell lines) to 136 +/- 21 (6 cell lines) with the first two treatments but with further exposures the lifespan increased to 182 +/- 15 (5 cell lines). MNNG had no effect when administered at the older age. Exposure of P. tetraurelia to 4-nitroquinoline-N-oxide at 0.021 mg/ml twice for 12 and 15 min at the younger age reduced the mean clonal lifespan from 143 +/- 28 to 125 +/- 21 and the maximum lifespan from 263 +/- 33 to 175 +/- 25.     

Analysis of the micronuclear B type surface protein gene in Paramecium tetraurelia.

The micronuclear DNA of Paramecium contains sequences that are precisely excised during the formation of the macronuclear (somatic) genome. In this paper we show that four eliminated sequences ranging in size from 28 to 416 base pairs, are present in or near the micronuclear copy of the B surface protein gene. Each excised sequence is bounded by the dinucleotide 5'-TdA-3'. Comparison of the micronuclear B gene with the previously determined micronuclear sequence of the A surface protein gene shows that although the positions of at least three of the eliminated sequences are conserved in both genes, the sequences are highly divergent. Transformation of vegetative macronuclei with fragments of the micronuclear B gene results in replication and maintenance of the DNA, but the micronuclear specific sequences are not removed. Previous studies have shown that the correct incorporation of the B gene into the new macronucleus requires copies of the macronuclear B gene in the old macronucleus. Using macronuclear transformation, we show that the micronuclear B gene can substitute for the macronuclear B gene with regard to its role in DNA processing. This suggests that the macronuclear DNA is not acting as a guide for the excision of the micronuclear specific sequences.     

A multigene family encoding R-SNAREs in the ciliate Paramecium tetraurelia

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane interactions and are conventionally divided into Q-SNAREs and R-SNAREs according to the possession of a glutamine or arginine residue at the core of their SNARE domain. Here, we describe a set of R-SNAREs from the ciliate Paramecium tetraurelia consisting of seven families encoded by 12 genes that are expressed simultaneously. The complexity of the endomembrane system in Paramecium can explain this high number of genes. All P. tetraurelia synaptobrevins (PtSybs) possess a SNARE domain and show homology to the Longin family of R-SNAREs such as Ykt6, Sec22 and tetanus toxin-insensitive VAMP (TI-VAMP). We localized four exemplary PtSyb subfamilies with GFP constructs and antibodies on the light and electron microscopic level. PtSyb1-1, PtSyb1-2 and PtSyb3-1 were found in the endoplasmic reticulum, whereas PtSyb2 is localized exclusively in the contractile vacuole complex. PtSyb6 was found cytosolic but also resides in regularly arranged structures at the cell cortex (parasomal sacs), the cytoproct and oral apparatus, probably representing endocytotic compartments. With gene silencing, we showed that the R-SNARE of the contractile vacuole complex, PtSyb2, functions to maintain structural integrity as well as functionality of the osmoregulatory system but also affects cell division.     

Ionic regulation of adenylate cyclase from the cilia of Paramecium tetraurelia.

The kinetics of the ionic regulation of an adenylate cyclase associated with the excitable ciliary membrane from Paramecium tetraurelia was examined. Glycerol (30%, v/v) stabilized the enzyme, and activated by an increase in Vmax. (3-fold) and a decrease in the apparent Km for MgATP (6-fold). Kinetic analysis of Mg2+ effects showed a stimulation via a single metal-binding site separate from the substrate site, with a dissociation constant, Ks, of 0.27 mM. Analysis of Ca2+ effects showed (i) an uncompetitive inhibition with respect to substrate MgATP, and (ii) dependence of the extent of inhibition on the free Mg2+ concentration. Ki values ranged from 4 to 130 microM-Ca2+ in the presence of 0.55-2 mM-Mg2+ respectively. This indicates competition between Mg2+ and Ca2+ at the metal-binding site. The Ca2+ effect was specific; Sr2+ and Ba2+ were almost without effect, and 100 microM-Ba2+ did not interfere with the Ca2+ inhibition. The actions of Ca2+ were readily reversible after addition of EGTA. K+ activated the adenylate cyclase at concentrations around 20 mM. The stimulatory potency of K+ was dependent on the free Mg2+ concentration. At 1 mM free Mg2+, 20 mM-K+ doubled the adenylate cyclase activity. The inhibitory Ca2+ and stimulatory K+ inputs were independent of each other.     

Mendelian and non-mendelian mutations affecting surface antigen expression in Paramecium tetraurelia.

A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium tetraurelia. Two of the mutations isolated by this procedure proved to be in modifier genes. The two genes are unlinked to each other and unlinked to the structural A i-antigen gene. These are the first modifier genes identified in a Paramecium sp. that affect surface antigen expression. Another mutation was found to be a deletion of sequences just downstream from the A i-antigen gene. In cells carrying this mutation, the A i-antigen gene lies in close proximity to the end of a macronuclear chromosome. The expression of the A i-antigen is not affected in these cells, demonstrating that downstream sequences are not important for the regulation and expression of the A i-antigen gene. A stable cell line was also recovered which shows non-Mendelian inheritance of a macronuclear deletion of the A i-antigen gene. This mutant does not contain the gene in its macronucleus, but contains a complete copy of the gene in its micronucleus. In the cytoplasm of wild-type animals, the micronuclear gene is included in the developing macronucleus; in the cytoplasm of the mutant, the incorporation of the A i-antigen gene into the macronucleus is inhibited. This is the first evidence that a mechanism is available in ciliates to control the expression of a gene by regulating its incorporation into developing macronuclei.     

A Novel Member of the Cyclin-Dependent Kinase Family in Paramecium tetraurelia

Passage through the cell cycle in eukaryotes requires the successive activation of different cyclin-dependent protein kinases. Here, we describe the identification and characterization of a novel class of cyclin-dependent protein kinase, termed Cdk2, in the ciliate Paramecium tetraurelia. It is 301 amino acids long, 7 amino acids shorter than Cdk1, the CDK that is associated with macronuclear DNA synthesis. All the catalytic domains typical of protein kinases can be located within the sequence and putative regulatory phosphorylation sites equivalent to Thr14, Tyr15, and Thr161 in human CDK1 are also conserved. The 'PSTAIRE' region characteristic of most CDKs is perfectly conserved. Cdk2 shares only 48% homology to Cdk1 at the amino acid level, suggesting that the evolutionary separation of Cdk1 and Cdk2 is ancient, and implying that they have different roles in cell cycle regulation. Like Cdk1, Cdk2 does not bind to yeast p13suc1, even though it has better conservation of p13suc1 binding sites than Cdk1 does. The Cdk2 protein level is relatively constant throughout the vegetative cell cycle. Cdk2 exhibits kinase activity towards bovine histone H1 in vitro with the maximal level late in the cell cycle, suggesting it may be involved in the regulation of cytokinesis. Our results further support the view that an analogue of the cyclin-dependent kinase cell cycle regulatory system like that of yeast and higher eukaryotic cells operates in Paramecium and that a family of cyclin-dependent kinases may control different aspects of the Paramecium cell cycle.     

Ultrastructure of the proximal region of somatic cilia in Paramecium tetraurelia

The morphology of the transition zone between the terminal plate of the basal body and the 9 + 2 region of the somatic (non-oral) cilium has been examined in Paramecium tetraurelia. Freeze-fracture and thin- section techniques disclosed both membrane specializations and various internal structural linkages. Freeze-fracture material revealed sets of particles interrupting the unit membrane. The more distal of these form plaquelike arrays while the proximal set of particles forms the ciliary "necklace." The plaque regions correspond to anionic sites on the outer membrane surface as revealed by binding of polycationic ferritin. Both the plaque particles and the necklace particles appear to be in contact with outer doublet microtubules via a complex of connecting structures. In the interior of the transition zone an axosomal plate supports an axosome surrounded by a ring of lightly packed material. Only one of the two central tubules of the axoneme reaches and penetrates the axosome. Below the axosomal plate four rings, each approx. 20 nm wide, connect adjacent outer doublets. An intermediate plate lies proximal to these rings, and a terminal plate marks the proximal boundary of this zone. Nine transitional fibers extend from the region of the terminal plate to the plasmalemma. The observations described above have been used to construct a three-dimensional model of the transition region of "wild-type" Paramecium somatic cilia. It is anticipated that this model will be useful in future studies concerning possible function of transition-zone specializations, since Paramecium may be examined in both normal and reversed ciliary beating modes, and since mutants incapable of reverse beating are available.     

AMP-dependence of the cyanide-insensitive pathway in the respiratory chain of Paramecium tetraurelia

The AMP-dependent stimulation of the cyanide-insensitive respiration of Paramecium mitochondria was investigated. The nucleotides exhibiting a stimulatory effect on the cyanide-insensitive oxidation of pyruvate (+ malate) in a medium supplemented with EDTA or carboxyatractyloside were, in decreasing order of efficiency, AMP, GMP, IMP, UMP and TMP. On the other hand, ADP, ATP and cyclic AMP were ineffective. In the presence of carboxyatractyloside, addition of AMP to Paramecium mitochondria incubated with pyruvate (+malate) led to an increase in membrane potential. In the absence of light, the photoactivable derivative of AMP, 3'-[4-[N-(4-azido-2-nitrophenyl)amino]butyryl]-AMP (NAP4-AMP) added to Paramecium mitochondria opposed the stimulatory effect of AMP on the cyanide-insensitive respiration; the Ki for NAP4-AMP was much lower than the Km for AMP, 0.2 microM compared with 120 microM. The ADP-stimulated respiration was not affected. Photoirradiation of Paramecium mitochondria in the presence of NAP4-AMP resulted in irreversible inhibition of the AMP-stimulated cyanide-insensitive respiration. No effect on the ADP-stimulated respiration was observed. A heatlabile cyanide-insensitive ubiquinol oxidase was extracted from Paramecium mitochondria with the detergent NN-dimethyl-N-(3-laurylamidopropyl)amine oxide. The quinol oxidase activity was slightly stimulated by AMP.