Protein Similarities in the Genus Tetrahymena and a Description of Tetrahymena leucophrys n. sp.1

The potential utility of protein arrays visualized by SDS-PAGE in taxonomic studies of protozoa has been investigated within the genus Tetrahymena. Matching coefficients have been obtained from one-dimensional separations of cytoskeletal proteins in a study involving 40 strains of Tetrahymena. In separate studies, the method was estimated to be accurate to within 10%, and the limit of intraspecific variation within the genus was estimated at ? 30%. Accordingly, it is suggested that strains showing matching coefficients 0.6 in these preparations probably represent different species. Further tests using strains identified to species by breeding criteria have shown that the 0.6 rule is asymmetric, i.e. although matching coefficients lower than this indicate separate species, values higher than this do not prove identity. Protein comparisons included in the present study indicate that strain Tur, previously identified as a strain of T. vorax, is distinct from all other macrostome-forming species. It is here designated Tetrahymena leucophrys n. sp.     

Variation In Surface Proteins In Tetrahymena*

SYNOPSIS. Surface proteins of Tetrahymena were identified by lactoperoxidase iodination, and comparisons were made between a number of strains and species within the genus. an adequate procedure for strain comparisons was found to be solubilization of whole cells following iodination, separation of total cell protein using polyacrylamide gel electrophoresis, and identification of surface proteins by autoradiography of dried gels. the results obtained in the present study show the existence of both interspecific and intraspecific variation in surface proteins of Tetrahymena, but the differences tend to be small within species and large between species. the relation of these cell surface fingerprints to the present taxonomic designations within the genus is discussed. Questions are raised about the functional significance of these surface proteins.     

Proteolytic degradation of ribosomal proteins during isolation of ribosomes and ribosomal subunits from Tetrahymena

A preparation procedure previously used to isolate active ribosomal subunits from an amicronucleate strain of Tetrahymena of undefined phenoset (T. «pyriformis CGL) yields inactive subunits when applied to other amicronucleate or to micronucleate strains of this protozoa.Proteolytic degradation of a small number of ribosomal proteins during preparation of ribosomal subunits from these strains explains this results. If cell extraction and ribosome isolation are carried out in the presence of iodoacetamide, proteolytic activity is inhibited and active ribosomal subunits are obtained. Comparison of the protein complements of active ribosomal subunits prepared in the presence of iodoacetamide from three amicronucleate strains of Tetrahymena reveals small but significant differences.     

An electrophoretic comparison of the histones of various strains of Tetrahymena pyriformis

Histones were extracted from isolated macronuclei of several strains of the ciliated protozoan Tetrahymena pyriformis and compared by electrophoresis on both urea-acrylamide and sodium dodecyl sulfate-acrylamide gels. High resolution urea-acrylamide-gel electrophoresis resolves Tetrahymena histones into five main classes. The lysine-rich histone H1 exhibits microheterogeneity within each strain, mostly due to phosphorylation, and varies extensively in electrophoretic mobility and apparent molecular weight among the strains. Both H3 and H4 are constant among Tetrahymena strains and consist of several secondarily modified subspecies. However, while the electrophoretic constancy of H4, observed in higher organisms, extends to this lower eukaryote, H3 of each Tetrahymena strain migrates faster than calf thymus H3 in both gel systems. This suggests that H3 is not as rigidly conserved as H4. Fraction HX has no electrophoretic counterpart in calf thymus histone. It consists of five subfractions, each of which displays a remarkably constant electrophoretic mobility among the various strains. H2B is electrophoretically variable among Tetrahymena strains. The intersyngen and interphenoset diversity of Tetrahymena histone is shown to be comparable to that found among vertebrate classes.     

Tetrahymena Tubulins and In Vitro Translation of Tetrahymena RNA*

SYNOPSIS. Tetrahymena outer doublet tubulin was compared with neurotubulin and Chlamydomonas flagellar tubulin on SDS-polyacrylamide gels. Tetrahymenaα tubulin did not comigrate with either brain or flagellar α tubulins, although brain, flagellar, and ciliary β tubulins all comigrated. Axonemal tubulin from Tetrahymena strain ST was compared with this tubulin from strains W. S. HSM, and E, and all were found to have the same mobilities. Poly-A containing RNA was separated from whole cell Tetrahymena RNA by oligo-dT cellulose chromatography. Poly-A+ RNA from 24-h cultures (early exponential growth) stimulated greater incorporation of amino acids into polypeptides in the wheat germ cell-free translation system than did poly-A+ RNA from 36-h and 49-h cultures. When separated on SDS-polyacrylamide gels, the translation products of the 24-h poly-A + RNA had 2 prominent protein bands which comigrated with α and β tubulin isolated from Tetrahymena cilia. These bands were not found in the translation products of poly-A+ RNA isolated from 49-h cultures or in the translation products ofpoly-A- RNA.     

THE TETRAHYMENA HOMOLOG OF BACTERIAL AND MAMMALIAN 4-HYDROXYPHENYLPYRUVATE DIOXYGENASES LOCALIZES TO MEMBRANES OF THE ENDOPLASMIC RETICULUM

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis ofTetrahymena HPPD takes place in cells starved for more than 30min, these results suggest that there is a flow ofTetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and thatTetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.     

Serotypic Variation Among Isolates of Ichthyophthirius multifiliis Based on Immobilization

Efforts have been made to determine whether surface antigens could be used as biochemical markers to define strain differences in the parasitic ciliate Ichthyophthirius multifiliis. In previous studies, a wild-type isolate designated G1 was found to have surface proteins analogous to the immobilization antigens of Paramecium and Tetrahymena; rabbit antiserum against this strain immobilizes homologous cells in vitro. It has now been shown for two additional Ichthyophthirius isolates (designated G1.1 and G2) that immobilization antigens are both present and serologically distinct. Proteins of similar size, which cross-react in Western blots with rabbit antisera against immobilization antigens of the G1 strain, are nevertheless found in the G1.1 and G2 isolates. As shown by Southern blotting analysis, the G1.1 and G2 strains also contain genomic DNA sequences which hybridize with an immobilization antigen cDNA from G1 when probed under conditions of reduced stringency. The serotypic differences in immobilization between I. multifiliis isolates appear to be stable over time and provide a means of discriminating strains. In addition to providing a basis for comparative studies, the work described here has implications for the development of vaccines against this important fish parasite.     

Characterization of the macronuclear DNA of different species ofTetrahymena

Summary The macronuclear DNAs from 20 different species ofTetrahymena were characterized using alternating Orthogonal Field (AOF) gel electrophoresis. Each species has approximately 300 different macronuclear DNA molecules that range in size from about 100–2000 kb pairs. Although the individual macronuclear DNA molecules are not well resolved on an AOF gel, most species have a unique profile of macronuclear DNA. The sequences that hybridize with histone H4 (Tetrahymena) and ubiquitin (yeast) genes were identified on the separated macronuclear DNA molecules of the different species. All species have 2 histone H4 genes located on macronuclear DNA molecules of different sizes. This is consistent with the duplication of the histone H4 gene prior to the speciation events leading to the various species ofTetrahymena. The number and sizes of the macronuclear DNA molecules that hybridize with the ubiquitin probe vary from species to species. A grouping of the different species ofTetrahymena based on this hybridization pattern paralels groupings of the species based on ribosomal RNA sequences and isoenzymes. Some intraspecific variation among different strains ofTetrahymena thermophila was detected using ubiquitin and 5S ribosomal RNA as probes.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

Low molecular weight DNA in the heteromeric macronuclei of two cyrtophorid ciliates

Summary— The size range of the native DNA molecules in the heteromeric macronuclei of two cyrtophorid ciliates (Trithigmostoma cucullulus, Chilodonella uncinata) was mainly investigated by using agarose gel electrophoresis. Numerous bands superimposed on a continuous spectrum of molecular sizes between about 0.35 kb and 30 kb were resolved by conventional electrophoresis. Species-specific banding patterns indicate a variation between species in the copy number of individual DNA fragments. A slight intra-specific variability of banding patterns can exist. Electrophoretic distributions for two strains of T cucullulus were indeed found to differ by at least one more intense band (‘overamplified’ sequences?). Fractionation by contour-clamped homogeneous electric field (CHEF) gel electrophoresis revealed that the size continum of macronuclear DNA molecules does not extend beyond 60–70 kb. The average size was estimated to be around 4 kb. Unresolved DNA fraction (> 1000 kb) accounted for less than 10% of the mass of cellular DNA entering CHEF gels. Macronuclear ribosomal DNA of each cyrtophorid species was identified by Southern hybridization with a Tetrahymena rDNA probe. The hybridization signal was observed on a single band of low molecular weight DNA. The corresponding size was close to 14.5 kb in Trithigmostoma and 15.5 kb in Chilodonella, which is about twice the size of monomeric rDNA in hypotrichous ciliates. We showed that S1 nuclease resistant duplexes wit half the length of the native rDNA can be formed by rapid renaturation of heat-denatured molecules and hybridized with native rDNA. This strongly suggests that the nucleotide sequence of this rDNA is a large palindrome. Unlike the hypotrichs, macronuclear rDNA in cyrtophorids should be organized into palindromic dimers as in Tetrahymena species.     

Microcalorimetric Study on the Toxic Effect of Pb<Superscript>2+</Superscript> to <Emphasis Type="Italic">Tetrahymena</Emphasis>

The toxic effect of Pb²⁺ has been studied in eukaryotic cells by using Tetrahymena as a target. The maximum power (P _m) and the growth rate constant (k) were determined, which showed that values of P _m and k were linked to the concentration (C) of Pb²⁺. The addition of Pb²⁺ caused a decrease of the maximum heat production and growth rate constant, indicating that Tetrahymena growth was inhibited in the presence of Pb²⁺, and Pb²⁺ took part in the metabolism of cells. From micrographs, morphological changes of Tetrahymena were observed with addition of Pb²⁺, indicating that the toxic effect of Pb²⁺ derived from destroying the membrane of surface of Tetrahymena. According to the thermogenic curves and photos of Tetrahymena under different conditions, it is clear that metabolic mechanism of Halobacterium halobium R1 growth has been changed with the addition of Pb²⁺.     

Electrophoretic Mobility Patterns of Ribosomal Proteins as an Aid to Phenoset Classification of Amicronucleate Strains of Tetrahymena1

Comparison of the electrophoretic migration patterns of proteins of active 40S and 60S ribosomal subunits isolated from nine amicronucleate strains of Tetrahymena of known phenoset revealed strain dependent differences which correlated with the phenoset classification of these strains as determined by Borden, Whitt & Nanney, who compared isoenzyme patterns.     

Functional comparision between truncated MTT1 and truncated MTT2 from Tetrahyemna thermophila

Metallothioneins (MTs) are low-molecular-weight proteins with high Cys content and high metal-chelating ability. CdMT and CuMT subfamilies present different characteristics in Tetrahymena. To explore the effect of the cysteine arrangement and sequence length of MTs for binding different metal ions, MTT1, truncated MTT1 (TM1), MTT2, and truncated MTT2 (TM2) were expressed in E. coli. The half-maximal inhibiting concentrations (IC₅₀) of Cd²⁺ and Cu⁺ for the recombinant strains were different. Furthermore, E. coli cells expressing MTT1 and TM1 exhibited higher accumulating ability for Cd²⁺ than cells expressing MTT2 and TM2. However, the opposite is true for Cu⁺. The binding ability of the different recombinant proteins to Cd²⁺ and Cu⁺ were also different. MTT1 and truncated mutant TM1 were the preference for Cd²⁺, whereas MTT2 and truncated mutant TM2 were the preference for Cu⁺ coordination. These results showed that metal ion tolerance and accumulation ability not only depended on cysteine arrangement pattern but also on sequence length of MT in Tetrahymena.     

Tetrin polypeptides are colocalized in the cortex of Tetrahymena

There is a complex system of 2- to 5-nm filaments in the oral apparatus of Tetrahymena. Four major subunit proteins, called tetrins, have been isolated from the filaments. These proteins, showing apparent molecular weights in polyacrylamide gels of 79-89 kDa, will assemble in vitro into 2- to 5-nm filaments. Tetrin filaments in vivo show different packing arrangements in different regions of the oral apparatus. We sought to determine the distributions of tetrin polypeptides within the complex oral structure by obtaining monoclonal antibodies specific for individual tetrins, then mapping their distributions within the oral apparatus using standard fluorescence microscopy, confocal laser scanning fluorescence microscopy, and electron microscopy. The results indicate that the four tetrin polypeptides are colocalized everywhere within the oral apparatus of Tetrahymena. Tetrin-binding proteins or specific nucleating structures may need to be invoked to explain the complex organization of the tetrin network. The 16 monoclonal antibodies obtained were also used to search for evidence of immunological relationships between tetrin and cytoskeletal proteins in multicellular organisms. None was found.     

Calyx fluid proteins of two Cotesia sesamiae (Cameron) (Hymenoptera: Braconidae) biotypes in Kenya: implications to biological control of the stem borer Busseola fusca (Fuller) (Lepidoptera: Noctuidae)

Cotesia sesamiae (Cameron) (Hymenoptera: Braconidae) is an indigenous larval endoparasitoid of Busseola fusca (Fuller) (Lepidoptera: Noctuidae) in sub-Saharan Africa. In Kenya, reports suggest that C. sesamiae occurs as two biotypes. Biotype avirulent to B. fusca gets encapsulated by haemocytes in this host and is unable to complete development. Biotype virulent to B. fusca is able to overcome immune defences. Factors present in the calyx fluid such as the PolyDNAviruses (PDV), venom and calyx fluid proteins have been implicated in the variation of C. sesamiae virulence against B. fusca. In the present study, calyx fluid proteins of the two C. sesamiae biotypes were compared using 2-D gel electrophoresis. More protein spots were observed in the virulent parasitoid calyx fluid, but some proteins were specifically observed in the avirulent parasitoid calyx fluid while others were observed in both. To study changes in proteins due to parasitism of B. fusca larvae by the two strains, SDS-PAGE gel were performed on fat body tissues and the haemolymph at three time points. Differences between the two strains were observed in both the fat body and haemolymph tissues. Parasitism-specific protein bands were detectable in fat body tissues of B. fusca larvae parasitized by the two C. sesamiae strains. These proteins were absent in unparasitized larvae. Implications for using C. sesamiae as a biocontrol agent of B. fusca in Africa are discussed.     

Genetic analysis of protein variations in Mus musculus using two-dimensional electrophoresis

We have investigated the variation of proteins from crude homogenates of mouse kidneys in several strains of Mus musculus by means of two-dimensional electrophoresis. In this study, we have used the strains C57BL/6J, DBA/2J, CD-1, M. m. castaneus, and M. m. molossinus, as well as offspring from crosses among these strains. Out of the 100 loci screened, we have found nine loci showing interstrain differences. We have been able to identify three proteins as Id-1, Car-2, and Sep-1. The remaining variants are probably new loci in the mouse. Most of the variants (seven) can be mapped to a chromosome. We have found also that differences in the protein pattern as seen on two-dimensional gels are small among subspecies of Mus musculus.     

Colpidium-Produced RNA as a Growth Stimulant for Tetrahymena*

SYNOPSIS. During logarithmic growth, Colpidium campylum produced a complex, heat-stable material which elevated the reproductive ate of Tetrahymena subsequently inoculated into the culture. This material stimulated only early stages in the growth of Tetrahymena—effect accompanied by an increased number and size of food vacuoles. The Colpidium product was found to be RNA, precipitable from the medium by acetone. Lipids and proteins or peptides were also present in the complex; these appeared to protect the RNA from the action of chemical and physical agents.     

The Purification of DNA from the Genomes of Paramecium aurelia and Tetrahymena pyriformis1

SYNOPSIS. Methods are described for the recovery of highly purified DNA from Paramecium aurelia and Tetrahymena pyriformis in high yields. Our DNA is only slightly contaminated with RNA and carbohydrate, and little or no protein can be detected. We could not reduce the RNA (orcinol-positive material) by further treatment (sephadex or hydroxyapatite chromatography and preparative CsCl gradients). At the extreme our DNA is contaminated with 15–20% RNA but the real value is most likely considerably lower than this. The DNA we have prepared from Paramecium and Tetrahymena shows all the properties of double-stranded, high molecular weight DNA when characterized by temperature melting, CsCl density gradient centrifugation and hydroxyapatite and sephadex chromatography. When denatured, it absorbs to nitrocellulose filters. The 2 major results of importance from our work reported here are: (1) There is similarity in base composition of DNA from different syngens of Paramecium (28% G+C for syngens 1, 2, 4, 5, and 9 and 29–30% G+C for syngen 8) while there is variation between the syngens of Tetrahymena (24–31% G+C for syngens 1, 4, 7, 10, 11, and 12); (2) the density of any Paramecium DNA varies depending upon whether the cells are grown in the presence of bacteria or in axenic medium. Our results are compatible with observations previously reported for Tetrahymena but contradict those made for Paramecium. The earlier reports of differences in base composition between syngens of Paramecium are probably due in large part to the use of stocks grown on bacteria.     

Cytoskeletal proteins of the cell surface in Tetrahymena : I. Identification and localization of major proteins

Isolated pellicles (cell ‘ghosts’) have been prepared from Tetrahymena thermophila strain B by two different methods. Using differential solubilization in combination with polyacrylamide gel electrophoresis and electron microscopy, we have tentatively identified the major proteins found in the surface-associated cytoskeleton. The ‘epiplasm’, a continuous layer of fibrous material found just beneath the surface membranes, appears to contain two major proteins. The smaller of the two (mol. wt 122 000 D) is believed to be present throughout the layer, whereas the larger protein (mol. wt 145 000 D) appears to be localized in the regions where ciliary basal bodies connect to the epiplasmic layer and to surface membranes. Evidence is presented which suggests that actin may also be present in this structure. Tubulin has been isolated from the cytosol of Tetrahymena and compared with cytoskeletal tubulin and porcine brain tubulin. A major protein of mol. wt 250 000 D which is found in Tetrahymena pellicles appears to be the major component of kinetodesmal fibers (striated elements which attach to the ciliary basal bodies).     

Evolution of the Response to Heat Shock in Genus Drosophila

Thermotolerance was studied in a wide spectrum of Drosophilaspecies and strains originating from different climatic zones and considerably differing from one another in the ambient temperature of their habitats. The species that lived in hot climate have a higher thermotolerance. Most species of the virilisgroup exhibited positive correlation between the HSP70 accumulation after heat exposure and thermotolerance; however, this correlation was absent in some species and strains. For example, the D. melanogasterOregon R strain, which had the highest sensitivity to heat shock (HS) among all strains and species studied, displayed the maximum level of HSP70 proteins after HS. The patterns of induction of various heat shock protein (HSP) families after heat exposure in a wide spectrum of Drosophila species were compared. The results obtained suggest that the HSP40 and low-molecular-weight HSPs (lmwHSPs) play a significant role in thermotolerance and adaptation to hot climate. Polymorphism in hsp70 gene clusters ofDrosophila and variation in the numbers of gene copies andhsp70 isoforms in group viriliswere found. The evolutionary role of the variation in the number of hsp70 gene copies observed in the strains and species of genusDrosophilais discussed.     

Insight into the core and variant exoproteomes of Listeria monocytogenes species by comparative subproteomic analysis

While Listeria monocytogenes is responsible for listeriosis, it is also a saprophytic species with exceptional survival aptitudes. Secreted proteins are one of the main tools used by bacteria to interact with their environment. In order to take into account the biodiversity of L. monocytogenes species, exoproteomic analysis was carried out on 12 representative strains. Following 2‐DE and MALDI‐TOF MS, a total of 151 spots were identified and corresponded to 60 non‐orthologous proteins. To categorize and analyze these proteomic data, a rational bioinformatic approach predicting final subcellular localization was carried out. Fifty‐two out of the 60 proteins identified (86.7%) were indeed predicted as localized in the extracellular milieu (gene ontology (GO): 0005576). Most of them (65.4%) were actually predicted as secreted via the Sec translocon. Comparative analysis allowed for proteins found in all or only in a subset of L. monocytogenes strains to be defined. While the core exoproteome included most proteins related to bacterial virulence, cell wall biogenesis, as well as proteins secreted by unknown pathways, a slight variation in the protein members of these categories were observed and constituted the variant exoproteome. This investigation resulted in the first definition of the core and variant exoproteomes of L. monocytogenes where corollaries on bacterial physiology are further discussed.