Tetrahymena tubulins and in vitro translation of Tetrahymena RNA.

Tetrahymena outer doublet tubulin was compared with neurotubulin and Chlamydomonas flagellar tubulin on SDS-polyacrylamide gels. Tetrahymena alpha tubulin did not comigrate with either brain or flagellar alpha tubulins, although brain, flagellar, and ciliary beta 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 alpha and beta 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 of poly-A- RNA.     

Increased levels of mRNAs for tubulin and other flagellar proteins after amputation or shortening of Chlamydomonas Flagella

A dramatic stimulation of synthesis of flagellar proteins occurs in Chlamydomonas following flagellar removal or experimentally induced resorption of the flagella into the cell. In this report we show that this stimulation involves an increase in the levels of mRNAs for tubulin and many other flagellar proteins. Total RNA and poly(A) RNA were isolated from cells after deflagellation or flagellar resorption, and were then translated in the reticulocyte lysate system. Two-dimensional gel analysis of the translation products demonstrates that the RNA-directed in vitro synthesis of α and β tubulins, and a number of other flagellar proteins, increases after deflagellation or flagellar resorption. Surprisingly, the α-tubulin synthesized in vitro does not co-migrate on two-dimensional gels with mature flagellar α-tubulin. Moreover, in vivo labeling experiments show that the major α-tubulin synthesized in the cell after deflagellation co-migrates with the major α-tubulin made in vitro, not with the major α-tubulin present in the flagella. These results suggest that flagellar α-tubulin is synthesized as a precursor, and undergoes post-translational modification before assembly into the flagella. In addition, we report that the synthesis of tubulin and other flagellar proteins can be specifically inhibited, as well as stimulated. Treatment of cells with IBMX, which induces flagellar resorption, causes a marked decrease in the levels of translatable mRNAs for tubulin and other flagellar proteins, without affecting levels of mRNAs for nonflagellar proteins.     

Tubulin Genes in the Algal Protist Euglena gracilis

ABSTRACT Alpha- and beta-tubulin cDNA were selected from a Euglenaλgt11 expression library, recloned and either sequenced (α-tubulin cDNA) or hybridized to Euglena RNA and DNA (α- and β-tubulin cDNA). RNA for hybridization was extracted at 30 minute intervals after flagellar amputation and quantitated for cDNA binding. Unlike previous reports on most other flagellates, no net increase in either α- or β-tubulin RNA could be detected during regeneration—suggesting steady state or constitutive tubulin RNA synthesis. Incubation of the cDNA with genomic DNA after restriction digestion produced patterns of hybridization consistent with the presence of one to two kinds each of the α- and β-tubulin genes. The deduced amino acid sequence of the α-tubulin cDNA was more than 90% identical to the α-tubulins of Trypanosoma, Chlamydomonas, Naegleria, Tetrahymena and higher plants. The carboxy terminus of the α-tubulin cDNA and the previously sequenced β-tubulin of Euglena showed greatest identity to the carboxy terminus of the tubulins from Trypanosoma brucei. The sequence data for α and β-tubulins of Euglena provides direct evidence for the similarity of two gene products from euglenas and trypanosomes and adds support to earlier suggestions that these organisms are phylogenetically related.     

Multiple tubulin forms in ciliated protozoan Tetrahymena and Paramecium species

Summary. Tetrahymena and Paramecium species are widely used representatives of the phylum Ciliata. Ciliates are particularly suitable model organisms for studying the functional heterogeneity of tubulins, since they provide a wide range of different microtubular structures in a single cell. Sequencing projects of the genomes of members of these two genera are in progress. Nearly all members of the tubulin superfamily (α-, β-, γ-, δ-, ɛ-, η-, θ-, ι-, and κ-tubulins) have been identified in Paramecium tetraurelia. In Tetrahymena spp., the functional consequences of different posttranslational tubulin modifications (acetylation, tyrosination and detyrosination, phosphorylation, glutamylation, and glycylation) have been studied by different approaches. These model organisms provide the opportunity to determine the function of tubulins found in ciliates, as well as in humans, but absent in some other model organisms. They also give us an opportunity to explore the mechanisms underlying microtubule diversity. Here we review current knowledge concerning the diversity of microtubular structures, tubulin genes, and posttranslational modifications in Tetrahymena and Paramecium species. Correspondence and reprints: Institute of Molecular Genetics, Czech Academy of Sciences, Vídeňská 1083, 14220 Prague, Czech Republic.     

BIOSYNTHESIS OF HETEROGENEOUS FORMS OF MAMMALIAN BRAIN TUBULIN SUBUNITS BY MULTIPLE MESSENGER RNAs

—Heterogeneity among the primary translation products of rat brain tubulin messenger RNA was examined. On two-dimensional gels native cytoplasmic tubulin from randomly bred rats (PB21) consists of two groups of α tubulin subunits among which the most acidic forms, α₁ and α₂, are most abundant; and β tubulin consists of a minimum of two species, β₁ and β₂. In the same group of animals the primary translation products of rat brain tubulin mRNA consist of at least these four subunit forms (α₁α₂, β₁ and β₂); however, minor basic forms of α subunits were not synthesized. This same result was obtained from a homologous brain protein synthesizing system, a heterologous system prepared from brain polysomes and rabbit reticulocyte initiation factors, and a wheat germ lysate programmed with brain poly A mRNA. A variant form of brain tubulin was found in rats bred monogamously for over 30 generations (MB71 rats). MB71 brain polysomes synthesize overlapping a subunits which migrate in two-dimensional gels to the α₁ position, and the typical PB21 α₂ is not present. The addition of PB21 brain mRNA to a protein synthesizing system composed of MB71 polysomes plus reticulocyte initiation factors allowed synthesis of the typical α₂ tubulin in addition to the MB71 tubulin subunits. The structural relationship among subunits was examined by radioiodinated peptide mapping. The α subunits are structurally different from the β subunits; however, among the major tyrosine-containing tryptic peptides no prominent differences were observed between α₁ and α₂, or between β₁ and β₂ by the radioiodination procedure. The results provide evidence for heterogeneity among the primary translation products of brain tubulin mRNA, and for the existence of multiple functional tubulin genes in rat brain.     

Tubulin evolution: Ciliate-specific epitopes are conserved in the ciliary tubulin of metazoa

Summary In spite of their overall evolutionary conservation, the tubulins of ciliates display electrophoretic and structural particularities. We show here that antibodies raised againstParamecium andTetrahymena ciliary tubulins fail to recognize the cytoplasmic tubulins of all the metazoans tested. Immunoblotting of peptide maps of ciliate tubulins reveals that these antibodies react with one or very few ciliate-specific epitopes, in contrast to polyclonal antibodies against vertebrate tubulins, which are equivalent to autoantibodies and recognize several epitopes in both ciliate and vertebrate tubulins. Furthermore, we show that the anti-ciliate antibodies recognize ciliary and flagellar tubulins of metazoans ranging from sea urchin to mammals (with the exception of humans). The results support the conclusion that although duplication and specialization of tubulin genes in metazoans may have led to distinct types of tubulins, the axonemal one has remained highly conserved.     

Microtubule proteins in axolotl eggs and developing embryos

Tubulin from eggs and embryos of the Mexican axolotl was characterized by electrophoresis and colchicine binding. In urea-polyacrylamide gel electrophoresis, soluble axolotl egg tubulin migrated as two bands, identical to tubulins from sea urchin sperm and Drosophila eggs. However, in SDS-containing gels, on which the α and β subunits of standard tubulins were well resolved, axolotl egg tubulin migrated as a single band with an apparent molecular weight of 53,500. The method of disruption of the eggs affected both yield of tubulin from vinblastine sulfate precipitates and stability of the colchicine binding activity. The colchicine binding activity of soluble tubulin from gently disrupted eggs was specific and of high affinity, with properties similar to those reported for other tubulins. The tubulin pool in unfertilized eggs was determined to be approximately 2 μg/egg; the level decreased 20% after initiation of cleavage and then remained constant through development to postneurula stages. The colchicine binding activity of soluble tubulin from embryos was much less stable than that of unfertilized eggs and decreased further during development. No differences were found in properties of tubulin from eggs of several strains of normally pigmented axolotls; however, tubulin from albino eggs showed slightly different properties in both electrophoresis and colchicine binding. The colchicine binding activity of soluble tubulin accounts for only half the total activity in axolotl eggs; they possess, in addition, a particulate nontubulin colchicine binding activity.     

Tubulin and actin synthesis during brain development

Chick brain proteins from 5- through 13-day embryos were labeled with l-[³⁵S]methionine for 30 min in vitro and analyzed by two-dimensional gel electrophoresis. Autoradiographs of the gels were scanned with a computer-coupled densitometer to measure the relative rates of protein synthesis. The actins and the tubulins were the most abundant proteins and had the highest rates of synthesis. β and γ actin were synthesized at constant rates throughout this period of development, but the rate of tubulin synthesis increased fourfold. Six α tubulins and two β tubulins were distinguished, and they were all synthesized at all times. The relative rates of synthesis of these forms changed with development in a complex pattern, but the stoichiometry of α:β remained 1:1.     

Induction of microtubule protein synthesis in Chlamydomonas reinhardi during flagellar regeneration

Flagellar regeneration in gametes of Chlamydomonas reinhardi is initiated within 15–20 min after flagellar amputation and proceeds at a rapid but decelerating rate until by 90 min flagellar outgrowth is 80–85% complete. Sufficient flagellar protein reserves exist in the cytoplasm to allow regeneration of flagella $\text{1}\text{3}\text{–}\text{1}\text{2}$ normal length. Nevertheless, in vivo labeling with ¹⁴C-amino acids shows that microtubule protein and other flagellar proteins are synthesized de novo during flagellar regeneration. To determine whether tubulin is synthesized continuously by gametic cells or whether its synthesis is induced as a consequence of deflagellation, we have isolated polyribosomes from deflagellated and control cells, and analyzed the proteins produced by these polyribosomes during in vitro translation. Two proteins of 53,000 and 56,000 molecular weight which co-migrate with flagellar and chick brain tubulin on SDS-polyacrylamide gels and which selectively co-assemble with chick brain tubulin during in vitro microtubule assembly are synthesized by polyribosomes (or polyadenylated mRNA) from deflagellated cells. No microtubule proteins can be detected in the translation products synthesized by polyribosomes (or mRNA) from control cells, clearly indicating that deflagellation results in the induction of tubulin synthesis.Kinetics of tubulin synthesis demonstrate that induction takes place immediately after deflagellation; polyribosomes bearing tubulin mRNA can be detected in the cytoplasm in as little as 15 min after removal of flagella. Maximal rates of tubulin synthesis occur between 45 and 90 min after deflagellation when approximately 14% of the protein being synthesized by the cell is tubulin. This estimate of tubulin synthesis based on in vitro translation data agrees well with in vivo measurements of flagellar tubulin synthesis. While high levels of tubulin production extend well beyond the period of rapid flagellar assembly, synthesis begins to decline after 90 min, and by 180 min after deflagellation only low levels of tubulin mRNA are detectable in polyribosomes.     

Cloning,Purification, and Polymerization of Capsicum annuum Recombinant α and β Tubulin

α and β tubulin genes were cloned from the Capsicum annuum leaves using rapid amplification of cDNA ends (RACE)-PCR. Nucleotide sequence analysis revealed that 1,353 bp Capsicum annuum α?β-tubulin (CAnm α?β-TUB) encodes a protein of 450 amino acids (aa) each. The recombinant α?β tubulin was overexpressed mainly as an inclusion body in Escherichia coli BL21 (DE3), upon induction with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and its content was as high as 50% of the total protein content. Effective fusion protein purification and refolding are described. The average yields of α and β tubulin were 2.0 and 1.3 mg/l of culture respectively. The apparent molecular weight of each tubulin was estimated to be 55 kDa by SDS-polyacrylamide gel electrophoresis (PAGE). The tubulin monomers were found to be assembly competent using a standard dimerization assay, and also retained antigenicity with anti-His/T7 antibodies. The purified tubulins were polymerized to microtubule-like structures in the presence of 2 mM guanosine 5′-triphosphate (GTP).     

Euplotes Cytoskeleton: Tubulin and Microtubular Systems in Interphase1,2

The Euplotes cytoskeleton obtained by treatment with Triton X-100 in a microtubule-stabilizing buffer has been studied by electron microscopy and SDS-polyacrylamide electrophoresis. An antiserum has been prepared using the tubulin band from preparative gels of Euplotes cytoskeletons as an antigen. This antiserum reacts with the tubulin from different ciliated protozoa but fails to recognize the vertebrate tubulin by immunoblotting. Immunoblotting studies have demonstrated a slower electrophoretic mobility for the α-tubulin of Euplotes and Oxytricha than that of Paramecium and Tetrahymena. A cytoplasmic microtubular network in Euplotes has been revealed by indirect immunoftuorescence using both an anti-α-tubulin monoclonal antibody directed against chick brain tubulin and an antiserum raised against the tubulin of Euplotes.     

Translation in vitro of Tetrahymena pyriformis polyadenylated mRNA. Identification of tubulin amongst the translated products and demonstration of its heterogeneity.

The capacity of poly(A)-containing RNA of the protozoan ciliate Tetrahymena pyriformis to direct the synthesis of proteins in vitro has been tested using two cell-free systems: a wheat germ extract and a rabbit reticulocyte lysate. The results obtained with these two systems are compared and the identification of alpha and beta tubulins among the products of protein synthesis in vitro, after separation by one-dimensional and two-dimensional electrophoresis, is described. By isoelectric focusing in polyacrylamide gels, each species of tubulin is resolved into several bands, suggesting that the main subunits are more heterogeneous than has been generally described. Poly(A)-containing RNA has also been fractionated on a 70% formamide/sucrose gradient and it is shown that alpha and beta tubulins are coded by separate mRNAs.     

Cell-free processing of the secreted globins of Chironomus thummi (Diptera)

Poly(A)⁺ RNA from fourth instar larvae of the insect Chironomus thummi was efficiently translated in vitro. Up to 10% of the total cell-free translation products was immunoprecipitable with antiserum specific for chironomid haemoglobins. Six immunoreactive preglobin products were resolved on SDS-17.5% polyacrylamide gels, and were cotranslationally processed by dog pancreas microsomes to 2 major bands that comigrated with authentic secreted globins.     

Tetrahymena Telomerase RNA levels increase during macronuclear development

Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)_n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000–40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2-to 5-fold at 9–15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells. © 1992 Wiley-Liss, Inc.     

In vitro assembly of microtubule proteins from myxamoebae of Physarum polycephalum

Microtubule protein of >95% purity has been isolated by self-assembly from concentrated cell extracts of myxamoebae of Physarum polycephalum. Ninety-eight percent of the amoebal microtubule protein was tubulin. Both a and β subunits of amoebal tubulin were different from neurotubulin α and β subunits, but very similar to those of Tetrahymena ciliary tubulin. The non-tubulin components, which co-purified with tubulin through three assembly cycles, were essential to microtubule formation and contained several polypeptides including some of apparent molecular weights 49000, 57000 and 59000. Purified amoebal microtubule protein formed microtubules on warming in the absence of glycerol which were cold- and Ca²⁺-labile. In vitro, microtubule assembly was inhibited by vinblastine, benzimidazole derivatives and griseofulvin, but not by 10^?4 M colchicine. Amoebal tubulin had a much lower affinity than neurotubulin for colchicine.     

Biochemical and Structural Analyses of Microtubules in the Pellicular Membrane of Leishmania tropica1

The structure of the major protein of the pellicular membrane of Leishmania tropica was investigated. This protein is composed of two polypeptides, of ca. 50,000 d molecular weight, that were found to cross-react immunologically with the α and β subunits of pig brain tubulin. The polypeptides and pig brain tubulin subunits were partially digested with S. aureus V8 protease, and the peptides obtained analysed by SDS-polyacrylamide gel electrophoresis. A comparison of the patterns showed that the β subunits of Leishmania and pig tubulin have very similar primary structures, while the α subunits have evolved divergently. These experiments demonstrate that the major polypeptides found in the pellicular membrane of L. tropica are α and β subunits of tubulin. Immuno-electron microscopy indicates that the tubulin is located in the microtubules associated with the pellicular membrane of Leishmania. Arrays of microtubules were prepared by nonionic detergent treatment of the cells and observed by electron microscopy after negative staining. Optical diffraction reveals a 5 nm spacing between protofilaments in the microtubule and a 4 nm axial periodicity corresponding to the tubulin subunits. The pitch of the shallow left-hand three-start helix is 12°. A distance of 47 nm separates each microtubule from the next. These data show that the dimensions and supramolecular organization of the tubulin subunits in the microtubules are identical in the pellicular membrane of L. tropica and in mammalian brain.     

Separation of α- and β-Globin Messenger RNAs

THE 10S RNA fraction of reticulocytes from various species contains the haemoglobin messenger RNA^1–4. When this 10S RNA fraction is added to a cell-free system derived from reticulocytes or Krebs II ascites cells, it directs the synthesis of α and β chains of haemoglobin^5–8. The α and β messenger RNA molecules contained in this fraction, however, have not yet been separated and identified. When reticulocyte. RNA of mouse is subjected to electrophoresis on 6% polyacrylamide gels, the 10S fraction contains two major bands and three minor bands⁹, suggesting that the major lOS RNA bands contain the messenger RNAs for the α- and β-globin chains.     

A Procedure for Comparing Growing and Non-growing Tetrahymena Cultures: Ribonucleotide Pools in Tetrahymena Starved for the Essential Amino Acid Histidine1

ABSTRACT A new starvation procedure permitted the study of early events in a protozoon's growth cycle. Growing cultures of Tetrahymena that differed from non-growing cultures by one variable were produced by adding histidine to cells deprived of that amino acid in an otherwise complete medium. Alterations of the nucleotide pools were examined in +His and in -His cultures in the period preceding RNA synthesis by cells in +His medium. High performance liquid chromatographic analysis provided a balance sheet for the difference in purine compounds in the two cultures. The change in rNTP levels occurred only when the cells were resuspended in a fresh medium and was not a function of cell density. These observations point to the presence of a factor(s) in the old medium that inhibits the energy charge increase in rNTP and in purine accumulation.