A new noncanonical nuclear genetic code: translation of UAA into glutamate

Deviant genetic codes reported in ciliates share the same feature: one (UGA) or two (UAR) of the three canonical stop codons are translated into one particular amino acid. In many genera, such as Oxytricha, Paramecium, and Tetrahymena, UAR codons are translated into glutamine. UGA is translated into cysteine in Euplotes or into tryptophan in Colpoda inflata and Blepharisma americanum. Here, we show that three peritrich species (Vorticella microstoma, Opisthonecta henneguyi, and Opisthonecta matiensis) translate UAA into glutamate and that at least UAA in O. matiensis is decoded through a mutant suppressor-like tRNA. This kind of genetic code has never been reported for any living organism. Phylogenetic analysis with alpha-tubulin sequences corroborates that peritrichs, peniculines (Paramecium), and hymenostomates (Tetrahymena) form a monophyletic group (class Oligohymenophorea). The differential translation (glu/gln) of UAR codons, the monophyly of the Oligohymenophorea, and the common evolutionary origin of glutamate and glutamine suggest that deviant genetic codes of present-day oligohymenophoreans could have the same origin.     

Sequence of the small subunit ribosomal RNA gene from the hypotrichous ciliate Euplotes aediculatus

We have determined the complete nucleotide sequence of the coding region of the small subunit rRNA gene of the hypotrichous ciliate Euplotes aediculatus. It is 1882 nucleotides long and contains several inserts not present in the small subunit rRNA genes of the hypotrichs Oxytricha nova and Stylonychia pustulata. A comparison of the sequences suggests that E. aediculatus is much less closely related to these other two hypotrichs than they are to each other. Although the gene sequence of E. aediculatus is drifting more rapidly than those of these other two species, its faster evolutionary clock is not enough to account for the degree of difference between them.     

RNA Homologies Between Protozoa

SYNOPSIS. Comparison of RNA molecules between certain protozoa using the technic of nucleic acid hybridisation revealed that there are complementary sequences for ribosomal RNA molecules in the genomes of such cells. Furthermore the genes for ribosomal RNA have been conserved during evolution in this group of organisms. On the other hand, RNA molecules from these protozoa which can be considered to be "messengers" show little in the way of sequence relationships. By utilising the technic of hybridisation it was found that Oxytricha can compete effectively against Paramecium ribosomal RNA for Tetrahymena DNA but the ribosomal RNA sequences of the latter could not compete completely against Paramecium ribosomal RNA for Oxytricha DNA. The result is interpreted to show that different ribosomal sequences were hybridising with each of the DNA samples from Tetrahymena and Oxytricha. A general interpretation of this result in terms of ribosome evolution is presented.     

Codon Usage in Tetrahymena and Other Ciliates

Codon usage in ciliates was examined by analyzing the coding regions of 22 ciliate genes corresponding to a total of 26, 142 nucleotides (8, 714 codons). It was found that Tetrahymena, Paramecium and the hypotrichs ( Oxytricha and Stylonychia ) differed in which synonymous codons were used most frequently by their genes. In fact, the codon choices in highly expressed Tetrahymena genes were more similar to those of yeast genes than those of Paramecium genes. The ciliates do not appear to have unusually strong biases in codon usage frequency when compared to other protists such as yeast. The analysis of the Tetrahymena genes indicated that genes which are highly expressed during normal cell growth have a stronger bias towards using the "preferred" codons than those expressed at lower levels during growth or for brief periods during processes such as conjugation. This conforms to what is found in other protists.     

The Size of Macronuclear DNA and Its Relationship to Models for Maintaining Genic Balance*†

Synopsis.
A considerable amount of evidence is now available to indicate that the DNA in the ciliates Oxytricha and Stylonychia undergoes fragmentation when the micronucleus forms a macronucleus. Some evidence suggests that fragmentation may also occur in Tetrahymena and possibly in Paramecium . It is shown that some regulatory or nonrandom segregational mechanism must operate during cell divisions to maintain genic balance in Tetrahymena . Both the hypothesis of macronuclear subunits and also a new hypothesis based on replicative control of DNA are capable of explaining the currently know biochemical, cytological, and genetic facts.     

Codon usage in Tetrahymena and other ciliates

Codon usage in ciliates was examined by analyzing the coding regions of 22 ciliate genes corresponding to a total of 26,142 nucleotides (8,714 codons). It was found that Tetrahymena, Paramecium and the hypotrichs (Oxytricha and Stylonychia) differed in which synonymous codons were used most frequently by their genes. In fact, the codon choices in highly expressed Tetrahymena genes were more similar to those of yeast genes than those of Paramecium genes. The ciliates do not appear to have unusually strong biases in codon usage frequency when compared to other protists such as yeast. The analysis of the Tetrahymena genes indicated that genes which are highly expressed during normal cell growth have a stronger bias towards using the "preferred" codons than those expressed at lower levels during growth or for brief periods during processes such as conjugation. This conforms to what is found in other protists.     

Sequence of the Small Subunit Ribosomal RNA Gene from the Hypotrichous Ciliate Euplotes aediculatus1

ABSTRACT. We have determined the complete nucleotide sequence of the coding region of the small subunit rRNA gene of the hypotrichous ciliate Euplotes aediculatus. It is 1882 nucleotides long and contains several inserts not present in the small subunit rRNA genes of the hypotrichs Oxytricha nova and Stylonychia pustulata. A comparison of the sequences suggests that E. aediculatus is much less closely related to these other two hypotrichs than they are to each other. Although the gene sequence of E. aediculatus is drifting more rapidly than those of these other two species, its faster evolutionary clock is not enough to account for the degree of difference between them.     

Alpha/beta inversion of the Euplotes and Oxytricha tubulins.

Immunoblotting studies, using a polyclonal antibody specific for the alpha-tubulin of ciliates and an anti-beta-tubulin monoclonal antibody, demonstrated that the tubulins of the ciliates Euplotes and Oxytricha show alpha/beta inversion although less accentuated than that observed in Paramecium and Tetrahymena. Results suggest that (a) the alpha/beta inversion may be a common characteristic within the Phylum Ciliophora; and (b) the electrophoretic behaviour of the alpha-tubulin may be useful for establishing evolutionary relatedness between the ciliates.     

Phylogenetic relationships among 28 spirotrichous ciliates documented by rDNA

The contiguous sequence of the SSU rDNA, ITS 1, 5.8S, ITS 2, and approximately 1370 bp at the 5(') end of the LSU rDNA was determined in 25 stichotrichs, one oligotrich, and two hypotrichs. Maximum parsimony, neighbor-joining, and quartet-puzzling analyses were used to construct individual phylogenetic trees for SSU rDNA, for LSU rDNA, and ITS 1+5.8S+ITS 2, as well as for all these components combined. All trees were similar, with the greatest resolution obtained with the combined components. Phylogenetic relationships were largely consistent with classical taxonomy, with notable disagreements. DNA sequences indicate that Oxytricha granulifera and Oxytricha longa are rather distantly related. The oligotrich, Halteria grandinella, is placed well within the order Stichotrichida. Uroleptus pisces and Uroleptus gallina probably belong to different genera. Holosticha polystylata (family Holostichidae) and Urostyla grandis (family Urostylidae) are rather closely related. These rDNA sequence analyses imply the need for some modifications of classical taxonomic schemes.     

Chemical Homologies Between Protozoa

SYNOPSIS. Nucleic acid hybridisation involving DNA samples was used to study relationships between various protozoa. The most hybridisation or nucleic acid homology was always in the homologous reaction between 2 DNA samples from the same source. It was concluded that all the protozoa tested have nucleotide sequences or genes in common with Paramecium aurelia. The ciliates could be placed in a sequence of decreasing homology relative to Paramecium: Tetrahymena, Colpidium, Stentor, Didinium, Dileptus, Blepharisma. Actinosphaerium had fewer sequences than any of the ciliates and the flagellate Euglena had the fewest sequences in common. The bacterium Aerobacter had none. Similar relationships were inferred from competitive hybridisation experiments; these relationships were also in general mirrored by morphologic relationships and overall G + C base compositions which ranged from 46% for Euglena to 32% for P. aurelia. These experiments, it is hoped, will contribute to studies on origins of the metazoa.     

The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata

We have determined the complete nucleotide sequence of the small- subunit ribosomal RNA genes for the ciliate protozoans Stylonychia pustulata and Oxytricha nova. The sequences are homologous and sufficiently similar that these organisms must be closely related. In a phylogeny inferred from comparisons of several eukaryotic small-subunit ribosomal RNAs, the divergence of the ciliates from the eukaryotic line of descent is seen to coincide with the radiation of the plants, the animals, and the fungi. This radiation is preceded by the divergence of the slime mold, Dictyostelium discoideum.      

Guanylyl cyclases with the topology of mammalian adenylyl cyclases and an N-terminal P-type ATPase-like domain in Paramecium, Tetrahymena and Plasmodium.

We cloned a guanylyl cyclase of 280 kDa from the ciliate Paramecium which has an N-terminus similar to that of a P-type ATPase and a C-terminus with a topology identical to mammalian adenylyl cyclases. Respective signature sequence motifs are conserved in both domains. The cytosolic catalytic C1a and C2a segments of the cyclase are inverted. Genes coding for topologically identical proteins with substantial sequence similarities have been cloned from Tetrahymena and were detected in sequences from Plasmodium deposited by the Malaria Genome Project. After 99 point mutations to convert the Paramecium TAA/TAG-Gln triplets to CAA/CAG, together with partial gene synthesis, the gene from Paramecium was heterologously expressed. In Sf9 cells, the holoenzyme is proteolytically processed into the two domains. Immunocytochemistry demonstrates expression of the protein in Paramecium and localizes it to cell surface membranes. The data provide a novel structural link between class III adenylyl and guanylyl cyclases and imply that the protozoan guanylyl cyclases evolved from an ancestral adenylyl cyclase independently of the mammalian guanylyl cyclase isoforms. Further, signal transmission in Ciliophora (Paramecium, Tetrahymena) and in the most important endoparasitic phylum Apicomplexa (Plasmodium) is, quite unexpectedly, closely related.     

Monovalent cation-induced structure of telomeric DNA: the G-quartet model

We have investigated the structures formed by oligonucleotides composed of two or four repeats of the telomeric sequences from Oxytricha and Tetrahymena. The Oxytricha four-repeat molecule (d(T4G4)4 = Oxy-4) forms structures with increased electrophoretic mobility in nondenaturing gels containing Na+, K+, or Cs+, but not in gels containing Li+ or no added salt. Formation of the folded structure results in protection of a set of dG's from methylation by dimethyl sulfate. Efficient UV-induced cross-links are observed in Oxy-4 and the related sequence from Tetrahymena (d(T2G4)4 = Tet-4), and join thymidine residues in different repeats. Models proposed to account for these data involve G-quartets, hydrogen-bonded structures formed from four guanosine residues in a square-planar array. We propose that the G-quartet structure must be dealt with in vivo by the telomere replication machinery.     

Molecular evolution in hypotrichous ciliates: Sequence of the small subunit ribosomal RNA genes fromOnychodromus quadricornutus andOxytricha granulifera (Oxytrichidae,Hypotrichida, Ciliophora)

Summary The small subunit ribosomal RNA (16S-like rRNA) coding regions of the hypotrichous ciliatesOnychodromus quadricornutus andOxytricha granulifera were amplified using polymerase chain reaction techniques. Complete sequences were determined for the amplified genes and compared to those of other ciliated protozoa. In phylogenetic trees inferred using distance matrix methods oxytrichids are not seen as a cohesive phylogenetic group.Oxytricha nova is most closely related toStylonychia pustulata in a lineage that also includesO. quadricornutus. This phylogeny contradicts phylogenetic schemes in whichOnychodromus is considered to be a primitive hypotrichous ciliate and suggests thatO. nova was misidentified as members of the genusOxytricha.     

Semi-automated recognition of protozoa by image analysis

A programme was created to semi-automatically analyse protozoal digitised images. Principal Component Analysis technique was used for species identification. After data collection and mathematical treatment, a three-dimensional representation was generated and several protozoa (Opercularia, Colpidium, Tetrahymena, Prorodon, Glaucoma and Trachelophyllum) species could be positively identified.     

Phylogenetic relationships within the class Oligohymenophorea,phylum Cilophora,inferred from the complete small subunit rRNA gene sequences ofColpidium campylum,Glaucoma chattoni,andOpisthonecta henneguyi

Summary Phylogenetic relationships within the class Oligohymenophorea, phylum Ciliophora, were investigated by determining the complete small subunit rRNA (SSrRNA) gene sequences for the hymenostomesColpidium campylum, Glaucoma chattoni, and the peritrichOpisthonecta henneguyi. The affiliations of the oligohymenophoreans were assessed using both distance matrix (DM) and maximum parsimony (MP) analyses. Variations do exist in the phylogenies created by the two methods. However, the basic tree topologies are consistent. In both the DM and MP analyses the hymenostomes (C. campylum, G. chattoni, and the tetrahymenas) all form a very tight group associated with the peritrichO. henneguyi. TheTetrahymena lineage was monophyletic whereasColpidium andGlaucoma were more closely related to each other than either was to the tetrahymenas. The monophyly of the genusTetrahymena in the present analysis supports the phylogenies determined from morphological data and molecular sequence data from the histone H3II/H4II region of the genome. The perplexing and controversial phylogenetic position of the peritrichs is once again depicted in the present analysis. The distinctiveness of the peritrichOpisthonecta from both hymenostome and nassophorean ciliates based on evolutionary distances suggests that the elevation of the peritrichs to a higher taxonomic rank should be reconsidered.     

Phylogenetic Relationships Among Hypotrichous Ciliates Determined with the Macronuclear Gene Encoding the Large, Catalytic Subunit of DNA Polymerase α

The complete macronuclear DNA polymerase α gene, previously sequenced in Oxytricha nova, has been cloned from a genomic macronuclear library and sequenced for the hypotrich O. trifallax. Macronuclear DNA clones of DNA polymerase α encoding ∼1000 amino acids, or approximately two-thirds of the open reading frame, have been obtained by PCR and sequenced for Halteria grandinella, Holosticha species, Paraurostyla viridis, Pleurotricha lanceolata, Stylonychia lemnae Teller, Sty. mytilus, Uroleptus gallina, and Urostyla grandis. Phylogenetic relationships inferred from DNA polymerase α amino acid sequences have been used to clarify taxonomic relationships previously determined by morphology of the cell cortex. Hypotrich phylogenies based on DNA polymerase α amino acid sequences are incongruent with morphological and other molecular phylogenies. Based upon these data, we assert that, contrary to morphological data, O. nova and O. trifallax are different species, and we propose that the oligotrich Halteria grandinella be reclassified as a hypotrich. This work also extends the available data base of eukaryotic DNA polymerase α sequences, and suggests new amino acid sequence targets for mutagenesis experiments to continue the functional dissection of DNA pol α biochemistry at the molecular level. Received: 7 January 1997 / Accepted: 7 April 1997     

Comparison of Sequence Differences in a Variable 23S rRNA Domain among Sets of Cryptic Species of Ciliated Protozoa

ABSTRACT Studies were undertaken to discover the relative molecular distances separating some familiar forms of ciliated protozoa, and the genetic species they include. Sequences of 190 bases of the D2 domain of the large ribosomal nucleic acid molecule were obtained by polymerase chain reaction from protists of three distinctive groups of ciliated protozoa- Colpoda, Paramecium and Tetrahymena. Evolutionary trees were constructed for each set of sequences using the PHYLOGEN 1.0 string programs. All three groups of ciliates manifested large molecular diversity among strains difficult or impossible to distinguish morphologically. The largest single evolutionary distance within a group was the 75 differences separating Tetrahymena paravorax from the other tetrahymenids. The largest mean distance for a group was the 21.2 for the colpodids. In all the protist groups the large molecular diversity is obscured by morphological conservatism associated with constraints of ancient designs. The molecular diversity within morphotypes argues for long evolutionary coexistence of species differentiated from each other in significant physiological, ecological, or nutritional ways.     

Immobilization of Ciliates by Concanavalin A*

SYNOPSIS. Interaction of several plant lectins with the ciliates Stylonychia mytilus, Euplotes aediculatus, and Tetrahymena pyriformis GL, was investigated. The motility of Stylonychia is specifically inhibited by treatment with concanavalin A, with which the 2 other ciliates react only weakly. Stylonychia can regain its motility by shedding the lectin-loaded surface components and rebuilding a new pellicle. Other lectins used in this study did not interact with these ciliates.