ABSTRACT
Tetrahymena pyriformis contains two arginine kinases, a 40-kDa enzyme (AK1) with a myristoylation signal sequence at the N-terminus and a two-domain 80-kDa enzyme (AK2). The former is localized mainly in cilia and the latter is in the cytoplasm. AK1 was successfully synthesized using an insect cell-free protein synthesis system and subjected to peptide mass fingerprinting (PMF) analysis. The masses corresponding to unmodified N-terminal tryptic peptide or N-terminal myristoylated peptide were not observed, suggesting that N-terminal peptides were not ionized in this analysis. We performed PMF analyses for two other phosphagen kinases (PKs) with myristoylation signals, an AK from Nematostella vectensis and a PK from Ectocarpus siliculosus. In both cases, the myristoylated, N-terminal peptides were clearly identified. The differences between the experimental and theoretical masses were within 0.0165-0.0583 Da, supporting the accuracy of the identification. Domains 1 and 2 of Tetrahymena two-domain AK2 were expressed separately in Escherichia coli and the extent of cooperativity was estimated on the basis of their kinetic constants. The results suggested that each of the domains functions independently, namely no cooperativity is displayed between the two domains. This is in sharp contrast to the two-domain AK from Anthopleura.
Subject(s)
Arginine Kinase/chemistry , Evolution, Molecular , Protozoan Proteins/chemistry , Tetrahymena pyriformis/chemistry , Amino Acid Sequence , Animals , Arginine Kinase/genetics , Arginine Kinase/metabolism , Cell-Free System/chemistry , Cell-Free System/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Molecular Weight , Peptide Mapping , Phaeophyceae/chemistry , Phaeophyceae/classification , Phaeophyceae/enzymology , Phaeophyceae/genetics , Phylogeny , Protein Structure, Tertiary , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sea Anemones/chemistry , Sea Anemones/classification , Sea Anemones/enzymology , Sea Anemones/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity , Tetrahymena pyriformis/classification , Tetrahymena pyriformis/enzymology , Tetrahymena pyriformis/geneticsABSTRACT
Domains of different evolutionary conservatism were defined in the 26S rDNA sequence of T. pyriformis. The fragment of studied DNA (1212 bp) showing high evolutionary conservatism was cloned. It was shown this fragment of DNA may be used to a probe for blot-hybridization analysis of the structure of rDNA from various taxa, protists to mammals. Superconservative and hypervariable domains were defined. The first are good for the primers for PCR analysis of rDNA from various taxa, the second--for species specific primers.
Subject(s)
Biological Evolution , DNA, Ribosomal/genetics , RNA, Ribosomal/genetics , Tetrahymena pyriformis/genetics , Animals , Cloning, Molecular , Humans , Nucleic Acid Conformation , Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Tetrahymena pyriformis/classificationABSTRACT
The 29 species of the Tetrahymena pyriformis complex are morphologically identical while being quite diverse at the molecular level. These species are also diverse relative to other eukaryotes. Phylogenetic relationships within the complex have been difficult to determine, because there are groups of closely related species, as well as individual species, that are highly divergent. We have sequenced portions of two highly conserved histone genes and the more rapidly evolving intergenic region between them. These sequences have been used to construct a phylogeny for the complex. A comparison of the amino-terminal portion of the histone H4 proteins from the species of the complex reveals a high degree of sequence diversity relative to that of other organisms. In contrast, the amino-terminal portions of the histone H3 proteins of the species in the complex are identical to each other. We also find that the pattern of nucleotide substitution in the intergenic region differs from that described for higher eukaryotes.
Subject(s)
Genetic Variation , Histones/genetics , Phylogeny , Tetrahymena pyriformis/genetics , Amino Acid Sequence , Animals , Biological Evolution , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Alignment/methods , Sequence Homology, Nucleic Acid , Tetrahymena pyriformis/classificationABSTRACT
Progress in ciliatology and in allied fields may demystify ciliate phylogenetics. Concentration on hymenostomes (mainly Tetrahymena and Paramecium) may have obscured directional features of ciliate physiology in phylogenetic problems. Therefore, means are suggested for "domesticating" the presumptively primitive, predominantly marine, sand-dwelling gymnostomes having nondividing macronuclei. The prize quarry is the marine psammophile Stephanopogon whose homokaryotic condition may mark it as a living fossil. Eventual axenic cultivation of these "primitive" ciliates may be aided by use as food of easily grown photosynthetic prokaryotes, some isolated from the marine sulfuretum or adjacent aerobic muds and sands where "karyorelictid" ciliates flourish.
