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.     

Crystal structure of AlgQ2, a macromolecule (alginate)-binding protein of Sphingomonas sp. A1 at 2.0A resolution

Sphingomonas sp. A1 possesses a high molecular mass (average 25,700 Da) alginate uptake system mediated by a novel pit-dependent ABC transporter. The X-ray crystallographic structure of AlgQ2 (57,200 Da), an alginate-binding protein in the system, was determined by the multiple isomorphous replacement method and refined at 2.0 A resolution with a final R-factor of 18.3% for 15 to 2.0 A resolution data. The refined structure of AlgQ2 was comprised of 492 amino acid residues, 172 water molecules, and one calcium ion. AlgQ2 was composed of two globular domains with a deep cleft between them, which is expected to be the alginate-binding site. The overall structure is basically similar to that of maltose/maltodextrin-binding protein, except for the presence of an N2-subdomain. The entire calcium ion-binding site is similar to the site in the EF-hand motif, but comprises a ten residue loop. This calcium ion-binding site is about 40 A away from the alginate-binding site.     

Motility Events of Trichocyst Insertion in Paramecium tetraurelia*

SYNOPSIS. Following electroshock-induced extrusion of its inserted trichocysts, Paramecium tetraurelia rapidly begins replacement of the population of lost organelles. Light microscopy of the cortical insertion of new trichocysts reveals a series of characteristic motility activities. An uninserted trichocyst in the cyclotic flow of the cell appears to be “captured” and removed to the noncyclotic, subcortical regions. The trichocyst then makes a series of saltatory motions which apparently serve to transport it to the cortex, with proper orientation (tip first) for insertion. Trichocyst saltations end with either cortical insertion of the organelle, or return to cyclosis. If the trichocyst is inserted, it makes a series of unique pivoting movements around the motionless tip. This form of motility, termed “wobble,” continues for a short period of time. After cessation of wobble, the insertion of the trichocyst is apparently complete, since no further motility is observed. With the aid of these observations it was possible to identify saltatory motility as the means for transporting trichocysts to the cortex for insertion, and also to observe a motility of unknown significance (wobble) apparently associated with the process of cortical insertion.     

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.     

Structure of porin refined at 1.8 A resolution.

The crystal structure of porin from Rhodobacter capsulatus has been refined using the simulated annealing method. The final model consists of all 301 amino acid residues well obeying standard geometry, three calcium ions, 274 solvent molecules, three detergent molecules and one unknown ligand modeled as a detergent molecule. The final crystallographic R-factor is 18.6% based on 42,851 independent reflections in the resolution range 10 to 1.8 A. The model is described in detail.     

A Role of Calcium Ions in Chemical Induction of Mating in Paramecium tetraurelia*

SYNOPSIS. Conjugation in Paramecium tetraurelia can be induced within mating-reactive cultures of a single mating type by treating the cells with solutions of KC1 + acriflavine in culture medium low in Ca²⁺. Gene mutations with known physiologic effect were used as selective inhibitors of cell surface membrane function to see which functions are necessary for chemical induction of conjugation. The results strongly suggest that a transient increase in the internal concentration of calcium at the very beginning of chemical induction is a necessary but not sufficient step.     

A mutation with pleiotropic effects on macronuclearly differentiated functions in Paramecium tetraurelia

The mtF^E mutation isolated in Paramecium tetraurelia affects mating type differentiation, trichocyst excretion, and viability. Its effect on mating type has already been shown to correspond to a restriction to the E mating type interpreted by an inefficiency of nuclear O-determining factors. In this paper we study the other two phenotypic characteristics whose hereditary transmission displays two unusual features. (1) In crosses between a wild-type strain and the mutant strain, the mutant characteristics do not reappear in F2 in the wild-type cytoplasmic lineage but only in F3 after the homozygous clones have undergone an additional nuclear reorganization. (2) Some F2 wild-type clones, in the mutant cytoplasmic lineage, retain some of the phenotypic characteristics of the mutant. We propose that the mtF gene product plays a role in the control of several macronuclearly differentiated functions.     

Studies on the Macronuclei of Doublet Paramecium tetraurelia: Distribution of Macronuclei and DNA at Fission*

SYNOPSIS. Doublet Paramecium tetraurelia would be expected to contain 2 macronuclei if their nuclear complement were strictly analogous to that of singlets. However, most doublets are unimacronucleate. It is shown in this study that dimacronucleate cells are present only in young clones. Unimacronucleate cells arise either through abnormalities in the determination and distribution of macronuclear anlagen during the first cell cycle after conjugation, or from dimacronucleate cells through abnormal division and segregation of macronuclei during the fission process. When a change in the number of macronuclei occurs through abnormalities in the division and segregation of daughter macronuclei, the daughter cells produced typically have DNA contents more similar than those expected from either random segregation of daughter macronuclei, or from the normal segregation pattern in ciliates in which changes in the number of macronuclei in progeny cells do not occur. This suggests that part of the regulation process of macronuclear DNA content in Paramecium may occur through control of the segregation pattern of daughter macronuclei.     

Structure of ubiquitin refined at 1.8 A resolution

The crystal structure of human erythrocytic ubiquitin has been refined at 1.8 A resolution using a restrained least-squares procedure. The crystallographic R-factor for the final model is 0.176. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.5 degrees, respectively. A total of 58 water molecules per molecule of ubiquitin are included in the final model. The last four residues in the molecule appear to have partial occupancy or large thermal motion. The overall structure of ubiquitin is extremely compact and tightly hydrogen-bonded; approximately 87% of the polypeptide chain is involved in hydrogen-bonded secondary structure. Prominent secondary structural features include three and one-half turns of alpha-helix, a short piece of 3(10)-helix, a mixed beta-sheet that contains five strands, and seven reverse turns. There is a marked hydrophobic core formed between the beta-sheet and alpha-helix. The molecule features a number of unusual secondary structural features, including a parallel G1 beta-bulge, two reverse Asx turns, and a symmetrical hydrogen-bonding region that involves the two helices and two of the reverse turns.     

A strange calmodulin of yeast

Calmodulin of Saccharomyces cerevisiae has different Ca2+ binding properties from other calmodulins. We previously reported that the maximum number of Ca2+ binding was 3 mol/mol and the fourth binding site was defective, which was different from 4 mol/mol for others. Their macroscopic dissociation constants suggested the cooperative three Ca2+ bindings rather than a pair of cooperative two Ca2+ bindings of ordinary calmodulin. Here we present evidence for yeast calmodulin showing the intramolecular close interaction between the N-terminal half domain and the C-terminal half domain, while the two domains of ordinary calmodulin are independent of each other. We will discuss the relationship of the shape and the shape change caused by the Ca2+ binding to the enzyme activation in yeast. The functional feature of calmodulin in yeast will also be considered, which might be different from the one of vertebrate calmodulin.     

Effects of an extremely low frequency electromagnetic field on the cell division rate and plasma membrane of Paramecium tetraurelia

The eukaryotic protozoan, Paramecium, was examined as a model for effects of pulsated electromagnetic fields (PEMF) on cells. A 72-Hz PEMF similar to fields employed clinically increased cell division rates in Paramecium by 8.5%. Two calcium transport mutants of these organisms showed differential responses to the same field. Verapamil, a calcium channel blocker, abolished any effect of PEMFs on cell division rates. A fluorescent probe that is thought to sense changes in membrane potential also manifested an altered response in the PEMF-exposed cells whereas a fluorescent lipid bilayer fluidity probe produced evidence of decreased membrane fluidity in the exposed cells. An effect of PEMFs on ion transport mediated by either a direct or indirect effect on the cell membrane is suggested by these studies.     

Axenic Paramecium caudatum. I. Mass Culture and Structure*

SYNOPSIS. To establish and grow Paramecium caudatum in mass axenic culture the culture medium of Soldo, Godoy & van Wagtendonk was modified by substituting phosphatidylethanolamine (PE) for TEM-4T and by a 10-fold increase in folic acid. Population densities of 4000 to 6000 cells/ml and a generation time of 20–26 h are regularly obtained. Optimal growth is obtained with PE-stigmasterol ratios between 40:1 to 400:1. Cells from 1-day-old axenic cultures have many lipid bodies aggregated in clumps (which disappear in 2 to 3 days) as well as foci of rough endoplasmic reticulum bordered by dictyosomes. The latter suggests a very active metabolism. Crystalline sheets found in both food vacuoles and lysosomes presumably play a role in digestion. Axenically grown cells also have abundant Golgi bodies (dictyosomes) and by late log phase become filled with lysosomes.     

Genetic Analysis of Trichocyst Discharge of the Wild Stocks of Paramecium tetraurelia*

SYNOPSIS. Aberrant discharge of trichocysts in response to picric acid occurs in 8 of the 28 wild stocks of Paramecium tetraurelia. There are at least 4 distinguishable phenotypes: nondischarge, stocks 139, 163, 169, and 242; temperature-sensitive nondischarge, stock 126; leaky nondischarge, stock 203; and a clonally unstable phenotype, stocks 146 and 148. From each of these stocks a single recessive gene causing nondischarge has been isolated by backcrosses to stock 51. The original stocks 126, 146, and 148 possess other genes which affect the extracted genes. The copper resistance locus is ～ 10 centiMorgans from nd169 and nd242, but none of the other nondischarge genes are linked to 6 marker loci. The genes nd169 and nd242 are only 0.5 centiMorgans apart making them the closest known pair of loci in P. tetraurelia. The genes nd126 and nd242 are distinguishable alleles at the same locus and the genes nd146 and nd148 are apparently identical alleles. The large number of loci involved in producing a similar phenotype in different stocks supports the idea that mutation is much more important than gene flow in this highly inbreeding species.     

Structure of calmodulin refined at 2.2 A resolution

The crystal structure of mammalian calmodulin has been refined at 2.2 A (1 A = 0.1 nm) resolution using a restrained least-squares method. The final crystallographic R-factor, based on 6685 reflections in the range 2.2 A less than or equal to d less than or equal to 5.0 A with intensities exceeding 2.5 sigma, is 0.175. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.7 degrees, respectively. The refined model includes residues 5 to 147, four Ca2+ and 69 water molecules per molecule of calmodulin. The electron density for residues 1 to 4 and 148 is poorly defined, and they are not included in the model. The molecule is shaped somewhat like a dumbbell, with an overall length of 65 A; the two lobes are connected by a seven-turn alpha-helix. Prominent secondary structural features include seven alpha-helices, four Ca2+-binding loops, and two short, double-stranded antiparallel beta-sheets between pairs of adjacent Ca2+-binding loops. The four Ca2+-binding domains in calmodulin have a typical EF hand conformation (helix-loop-helix) and are similar to those described in other Ca2+-binding proteins. The X-ray structure determination of calmodulin shows a large hydrophobic cleft in each half of the molecule. These hydrophobic regions probably represent the sites of interaction with many of the pharmacological agents known to bind to calmodulin.     

Fine Structure of the Contractile Vacuole Pore in Paramecium*

The pore through which a Paramecium contractile vacuole communicates with the external environment is a 1.2 μm long and 1 μm diameter cylindrical orifice in the pellicle. During diastole, the vacuole:pore junction is closed by a substantial diaphragm which parts to the side at systole. The diaphragm is composed of inner and outer membranes continuous with the vacuole and pore membranes, respectively, and an intervening cytoplasmic layer containing filaments and irregular membranous tubules and vesicles. Microtubules, organized into 2 sets, are an important component of the pore apparatus. One set of ～ 16 microtubules forms an annulus around the pore. These microtubules are organized into a right-handed helix with a pitch of 0.5-0.6 μm, and thus complete slightly more than 2 turns in their course from the level of the diaphragm to the pore outer lip. They appear to be embedded in a layer of dense material immediately adjacent to the pore membrane. The other set consists of 5 or more bands of 10–20 microtubules which radiate in a slight left-handed helix from an insertion at the pore out over the vacuole surface to the ampullae.     

Structure and refinement of penicillopepsin at 1.8 A resolution

Penicillopepsin, the aspartyl protease from the mould Penicillium janthinellum, has had its molecular structure refined by a restrained-parameter least-squares procedure at 1.8 Å resolution to a conventional R-factor of 0.136. The estimated co-ordinate accuracy for the majority of the 2363 atoms of the enzyme is better than 0.12 Å. The average atomic thermal vibration parameter, B, for the atoms of the enzyme is 14.5 Ų. One determining factor of this low average B value is the large central hydrophobic core, in which there are two prominent clusters of aromatic residues, one of nine, the other of seven residues. The N and C-terminal domains of penicillopepsin display an approximate 2-fold symmetry: 70 residue pairs are topologically equivalent, related by a rotation of 177 ° and a translation of 1.2 Å. The analysis of the secondary structural features of the molecule reveals non-linear hydrogen bonding. In penicillopepsin, there is no difference in the mean hydrogen-bond parameters for the elements of α-helix, parallel or antiparallel β-pleated sheet. The mean values for these structural elements are: NO, 2.90 Å; NHO, 1.95 Å; N?O, 160 °. The average hydrogen-bond parameters of the reverse β-turns and the 3₁₀ helices are distinctly different from the above values. The analysis of sidechain conformational angles χ¹ and χ² penicillopepsin and other enzyme structures refined in this laboratory shows much narrower distributions as compared with those compiled from unrefined protein structures. The close proximity of the carboxyl groups of Asp33 and Asp213 suggests that they share a proton in a tight hydrogen-bonded environment (Asp33OD2 to Asp213OD1 is 2.87 Å). There are several solvent molecules in the active site region and, in particular, O39 forms hydrogen-bonded interactions with both aspartate residues. The disposition of the two carboxyl groups suggests that neither is likely to be involved in a direct nucleophilic attack on the scissile bond of a substrate. The average atomic B-factors of the residues in this region of the molecule are between 5 and 8 Ų, confirming the proposal that conformational mobility of the active site residues has no role in the enzymatic mechanism. However, conformational mobility of neighbouring regions of the molecule e.g. the “flap” containing Tyr75, is verified by the high B-factors for those residues. The positions of 319 solvent sites per asymmetric unit have been selected from difference electron density maps and refined. Thirteen have been classified as internal, and several of these may have key roles during catalysis. The positively charged N^ζ atom of Lys304 forms hydrogen bonds to the carboxylate of Asp14 (internal ion pair) and to two internal water molecules O5 and O25. The protonated side-chain of Asp300 forms a hydrogen bond to Thr214O, 2.78 Å, and is the recipient of a hydrogen bond from a surface pocket water molecule O46. There is no possibility for direct interaction between Asp300 and Lys304 without large conformational changes of their environment. The intermolecular packing involves many protein-protein contacts (66 residues) with a large number of solvent molecules involved in bridging between polar residues at the contact surface. The penicillopepsin molecules resemble an approximate hexagonal close-packing of spheres with each molecule having 12 “nearest” neighbours.     

A model for the calmodulin-peptide complex based on the troponin C crystal packing and its similarity to the NMR structure of the calmodulin-myosin light chain kinase peptide complex.

In the crystal structure of troponin C, the holo C-domain is bound in a head-to-tail fashion to the A-helix of the apo N-domain of a symmetry-related molecule. Using this interaction, we have proposed a model for the calmodulin-peptide complex. We find that the interaction of the C-domain with the A-helix is similar to that observed in the NMR structure of the calmodulin-myosin light chain kinase (MLCK) peptide complex. This similarity in binding has enabled us to make a precise sequence alignment of the target peptides in the calmodulin-binding cleft and to rationalize the amino acid sequence-dependent binding strengths of various peptides. Our model differs from that proposed by Strynadka and James (Proteins Struct. Funct. Genet. 7, 234-248, 1990) in that the peptides are rotated by 100 degrees in the calmodulin binding cleft.     

Regulation of Macronuclear DNA Content in Paramecium tetraurelia*†

Synopsis.
The amitotic division of the macronucleus of Paramecium tetraurelia produces daughter macronuclei which frequently differ in DNA content. In wild-type cells these differences are small, but can be increased substantially by the action of mutant genes. The variance in macronuclear DNA content would increase continuously if there were no mechanism to regulate it. Paramecium has a very effective regulatory mechanism—all cells synthesize similar amounts of macronuclear DNA, regardless of the number of macronuclei or their prereplication DNA content. DNA synthesis is controlled at the level of macronuclear subunits, and the postreplication macronucleus consists of a mosaic of subunits that have undergone different numbers of replication events during the previous cell cycle. It is evident from experimental results that the amount of DNA synthesized can be influenced by the total size or mass of the cell. Experimental modification of the initial DNA content leads to no change in the amount of DNA synthesized, or in the subsequent protein content of the cells, but modification of cell size causes corresponding changes in the amount of DNA synthesized and in the size of the macronucleus. The implications of these observations for cell growth and the cell cycle are discussed.     

The crystal structure of staphylococcal nuclease refined at 1.7 A resolution

The crystal structure of staphylococcal nuclease has been determined to 1.7 A resolution with a final R-factor of 16.2% using stereochemically restrained Hendrickson-Konnert least-squares refinement. The structure reveals a number of conformational changes relative to the structure of the ternary complex of staphylococcal nuclease 1,2 bound with deoxythymidine-3',5'-diphosphate and Ca2+. Tyr-113 and Tyr-115, which pack against the nucleotide base in the nuclease complex, are rotated outward creating a more open binding pocket in the absence of nucleotide. The side chains of Ca2+ ligands Asp-21 and Asp-40 shift as does Glu-43, the proposed general base in the hydrolysis of the 5'-phosphodiester bond. The significance of some changes in the catalytic site is uncertain due to the intrusion of a symmetry related Lys-70 side chain which hydrogen bonds to both Asp-21 and Glu-43. The position of a flexible loop centered around residue 50 is altered, most likely due to conformational changes propagated from the Ca2+ site. The side chains of Arg-35, Lys-84, Tyr-85, and Arg-87, which hydrogen bond to the 3'- and 5'-phosphates of the nucleotide in the nuclease complex, are unchanged in conformation, with packing interactions with adjacent protein side chains sufficient to fix the geometry in the absence of ligand. The nuclease structure presented here, in combination with the stereochemically restrained refinement of the nuclease complex structure at 1.65 A, provides a wealth of structural information for the increasing number of studies using staphylococcal nuclease as a model system of protein structure and function.