Expression of mutated Paramecium telomerase RNAs in vivo leads to templating errors that resemble those made by retroviral reverse transcriptase.
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Telomeric DNA consists of short, tandemly repeated sequences at the ends of chromosomes. Telomeric DNA in the ciliate Paramecium tetraurelia is synthesized by an error-prone telomerase with an RNA template specific for GGGGTT repeats. We have previously shown that misincorporation of TTP residues at the telomerase RNA templating nucleotide C52 accounts for the 30% GGGTTT repeats randomly distributed in wild-type telomeres. To more completely characterize variable repeat synthesis in P. tetraurelia, telomerase RNA genes mutated at C52 (A, U, and G) were expressed in vivo. De novo telomeric repeats from transformants indicate that the predominant TTP misincorporation error seen in the wild-type telomerase is dependent on the presence of a C residue at template position 52. Paradoxically, the effects of various other telomerase RNA template and alignment region mutations on de novo telomeres include significant changes in fidelity, as well as the synthesis of aberrant, 5-nucleotide telomeric repeats. The occurrence of deletion errors and the altered fidelity of mutated P. tetraurelia telomerase, in conjunction with misincorporation by the wild-type enzyme, suggest that the telomerase RNA template domain may be analogous to homopolymeric mutational hot spots that lead to similar errors by the human immunodeficiency virus proofreading-deficient reverse transcriptase. (+info)
A Sec7-related protein in Paramecium.
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We have cloned and sequenced a SEC7-related gene in Paramecium tetraurelia that contains an open reading frame for 1135 amino acids encoding a 133 kDa protein, PSec7. Sec7, first identified in vesicular trafficking mutants in yeast, and its phylogenetic homologues function as guanine-nucleotide exchange factors for small G-proteins such as ARF (ADP-ribosylation factor). The deduced amino acid sequence in PSec7 for the motifs that form the ARF binding site are more than 70% identical to yeast Sec7 and similarly identical to ARNO, the human ARF exchange factor, with correct positioning of the critical glutamic acid residue within the motif region. Overall, the identity of PSec7 to yeast Sec7 is 32%. The deduced amino acid sequence also has five sequences that resemble IQ motifs, EF hand binding domains found in all myosins, and two pleckstrin homology domains. Similar sequences are present in yeast Sec7 and other Sec7-related molecules. A protein kinase A phosphorylation site may also be present. Southern blots suggest that a single gene encodes PSec7. Northern blots show that the message encoding PSec7 is induced on deciliation, followed by ciliogenesis, which suggests a role for PSec7 in cilia such as transport or targeting of ciliary membrane components. (+info)
Does ribosomal DNA get out of the micronuclear chromosome in Paramecium tetraurelia by means of a rolling circle?
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The macronuclear genes coding for rRNA (ribosomal DNA [rDNA]) of Paramecium tetraurelia, stock 51, are arranged in polymers consisting of units made up of a transcribed coding region and a nontranscribed spacer region. The whole macronuclear polymer ends with a portion of the spacer on either end followed by a telomere. Six kinds of macronuclear units, or genes, were mapped. Spacers were different, and transcribed regions were the same. These genes are found in markedly different numbers in the macronucleus. The most common gene shows two regions in the spacer where a sequence is followed by a direct repeat. The next most common gene is similar but shows a deletion plus a number of base pair substitutions. Although most cosmid clones contain only a single kind of gene, many contain more than one. These are thought to be produced by somatic crossing over. The four micronuclear genes that have been isolated consist of a single central transcribed region and portions of the spacer on either end. Sequencing indicates that the two ends of the molecule are partially redundant. While the spacer region at the right end of the macronuclear polymer is derived from the micronuclear spacer on the right, the spacer at the left end of the macronuclear polymer is derived from regions of the micronuclear spacer on both the right and the left. To account for this situation, a rolling-circle model for generation of the macronuclear rDNA from the micronuclear DNA is proposed. (+info)
Analysis of the three-dimensional trajectories of organisms: estimates of velocity, curvature and torsion from positional information.
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Most biological motions are three-dimensional. This includes the trajectories of whole organisms and of their appendages. While recordings of three-dimensional trajectories are sometimes published, quantitative analysis of these trajectories is uncommon, primarily because there are no standard techniques or conventions in biology for the analysis of three-dimensional trajectories. This paper describes a new technique, finite helix fit (FHF), based on the geometry of three-dimensional curves, whereby a three-dimensional trajectory is completely described by its velocity, curvature and torsion. FHF estimates these parameters from discretely sampled points on a trajectory (i.e. from positional data such as x,y,z coordinates). Other measures of motion can be derived from these parameters, such as the translational and rotational (or angular) velocities of an organism. The performance of the algorithms is demonstrated using simulated trajectories and trajectories of freely swimming organisms (a flagellate, Chlamydomonas reinhardtii; a ciliate, Paramecium tetraurelia; spermatozoa of a sea urchin, Arbacia punctulata; larvae of an ascidian, Botrylloides sp.). (+info)
Physical and physiological components of the graviresponses of wild-type and mutant Paramecium Tetraurelia.
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Wild-type and the morphological mutant kin 241 of Paramecium tetraurelia showed improved orientation away from the centre of gravity (negative gravitaxis) when accelerations were increased from 1 to 7 g. Gravitaxis was more pronounced in the mutant. A correlation between the efficiency of orientation and the applied g value suggests a physical basis for gravitaxis. Transiently enhanced rates of reversal of the swimming direction coincided with transiently enhanced gravitaxis because reversals occurred more often in downward swimmers than in upward swimmers. The results provide evidence of a physiological modulation of gravitaxis by means of the randomizing effect of depolarization-dependent swimming reversals. Gravity bimodally altered propulsion rates of wild-type P. tetraurelia so that sedimentation was partly antagonized in upward and downward swimmers (negative gravikinesis). In the mutant, only increases in propulsion were observed, although the orientation-dependent sensitivity of the gravikinetic response was the same as in the wild-type population. Observed swimming speed and sedimentation rates in the wild-type and mutant cells were linearly related to acceleration, allowing the determination of gravikinesis as a linear (and so far non-saturating) function of gravity. (+info)
Chemosensory signal transduction in paramecium.
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Paramecia are ciliated single-cell eukaryotic organisms that can respond to chemical cues in their environment. Glutamate is among those cues, which attract cells. We describe briefly here the following attributes of glutamate chemoresponse: 1) Cells are attracted to L-glutamate relative to KCl at high concentrations of glutamate. 2) There are at least two specific, relatively low affinity glutamate binding sites on the cell surface. Glutamate can be displaced from only one of the binding sites by inosine monophosphate (IMP), and quisqualate displaces glutamate from the second site, which is likely to be the glutamate receptor involved in attraction to glutamate. 3) IMP is a repellent and does not act synergistically with glutamate, whereas guanosine monophosphate (GMP) does. 4) Similarly, glutathione is an attractant, but glutamate and glutathione appear to use different transduction pathways. 5) Glutamate hyperpolarizes the cell. The ionic mechanism is not yet verified, but is likely to involve a K conductance. 6) Glutamate induces a rapid and robust increase in cAMP in the cell. Protein kinase A (PKA) is possibly involved in the transduction pathway because kinase inhibitors such as H7 and H8 inhibit glutamate response, but do not affect responses to other attractants, such as acetate and ammonium. Activation of PKA by the rapid rise in cAMP may sustain the hyperpolarization phosphorylation and activation of the plasma membrane calcium pump. 7) Candidate glutamate binding proteins are being identified among the cell surface proteins with the use of affinity chromatography. (+info)
Regulation of secretory protein gene expression in paramecium role of the cortical exocytotic sites.
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In cells that possess a regulated secretory pathway, exocytosis can lead to transcriptional activation of genes encoding products stored in secretory granules as well as genes required for granule biogenesis. With the objective of understanding this response, we have examined the expression of Paramecium secretory protein genes in different physiological and genetic contexts. The genes belong to the trichocyst matrix protein (TMP) multigene family, encoding polypeptides that form the crystalline matrix of the secretory granules, known as trichocysts. Approximately 1000 trichocysts per cell are docked at pre-formed cortical exocytotic sites. Their rapid and synchronous exocytosis can be triggered by vital secretagogues such as aminoethyldextran without harming the cells. Using this exocytotic trigger, we found that the transcription of TMP genes undergoes rapid, transient and co-ordinate 10-fold activation in response to massive exocytosis, leading to a 2.5-fold increase in the pool of TMP mRNA. Experiments with exocytosis-deficient mutants show that the secretagogue-induced increase in intracellular free calcium implicated in stimulus/secretion coupling is not sufficient to activate TMP gene expression. We present evidence that the state of occupation of the cortical exocytotic sites can affect TMP gene expression and suggest that these sites play a role in gene activation in response to exocytosis. (+info)
The cloning and molecular analysis of pawn-B in Paramecium tetraurelia.
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Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca(2+) current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca(2+) current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5' neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed. (+info)