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K Gull

Publications and source records attributed to K Gull.

At least 109 records · Page 6Linked to original sources
PubMed1987

Acetylated alpha-tubulin in Physarum: immunological characterization of the isotype and its usage in particular microtubular organelles.

We have used monoclonal antibodies specific for acetylated and unacetylated alpha-tubulin to characterize the acetylated alpha-tubulin isotype of Physarum polycephalum, its expression in the life cycle, and its localization in particular microtubular organelles. We have used the monoclonal antibody 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) as the probe for acetylated alpha-tubulin and have provided a biochemical characterization of the monoclonal antibody KMP-1 as a probe for unacetylated tubulin in Physarum. Concomitant use of these two probes has allowed us to characterize the acetylated alpha-tubulin of Physarum as the alpha 3 isotype. We have detected this acetylated alpha 3 tubulin isotype in both the flagellate and in the myxameba, but not in the plasmodium. In the flagellate, acetylated tubulin is present in both the flagellar axonemes and in an extensive array of cytoplasmic microtubules. The extensive arrangement of acetylated cytoplasmic microtubules and the flagellar axonemes are elaborated during the myxameba-flagellate transformation. In the myxameba, acetylated tubulin is not present in the cytoplasmic microtubules nor in the mitotic spindle microtubules, but is associated with the two centrioles of this cell. These findings, taken together with the apparent absence of acetylated alpha-tubulin in the ephemeral microtubules of the plasmodium suggest a natural correspondence between the presence of acetylated alpha-tubulin and microtubule organelles that are intrinsically stable or cross-linked.

Acetylation↗
PubMed1987

Subpellicular and flagellar microtubules of Trypanosoma brucei brucei contain the same alpha-tubulin isoforms.

The cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei brucei essentially consists of two microtubule-based structures: a subpellicular layer of singlet microtubules, which are in close contact with the cell membrane, and the flagellar axoneme. In addition, the cells contain a small pool of soluble tubulin. Two-dimensional gel electrophoretic analysis of the tubulins present in these subcellular compartments revealed two distinct electrophoretic isoforms of alpha-tubulin, termed alpha 1 and alpha 3. alpha 1-Tubulin most likely represents the primary translation product, while alpha 3-tubulin is a posttranslationally acetylated derivative of alpha 1-tubulin. In the pool of soluble cytoplasmic tubulin, alpha 1 is the predominant species, while the very stable flagellar microtubules contain almost exclusively the alpha 3-tubulin isoform. The subpellicular microtubules contain both isoforms. Neither of the two alpha-tubulin isoforms is organelle specific, but the alpha 3 isoform is predominantly located in stable microtubules.

Acetates↗
PubMed1987

Distinct localization and cell cycle dependence of COOH terminally tyrosinolated alpha-tubulin in the microtubules of Trypanosoma brucei brucei.

alpha-Tubulin can be posttranslationally modified in that its COOH-terminal amino acid residue, tyrosine, can be selectively removed and replaced again. This reaction cycle involves two enzymes, tubulin carboxypeptidase and tubulin tyrosine ligase. The functional significance of this unusual modification is unclear. The present study demonstrates that posttranslational tyrosinolation of alpha-tubulin does occur in the parasitic hemoflagellate Trypanosoma brucei brucei and that posttranslational tyrosinolation can be detected in both alpha-tubulin isoforms found in this organism. Trypanosomes contain a number of microtubular structures: the flagellar axoneme; the subpellicular layer of singlet microtubules which are closely associated with the cell membrane; the basal bodies; and a cytoplasmic pool of soluble tubulin. Tyrosinolated alpha-tubulin is present in all these populations. However, immunofluorescence studies demonstrate a distinct localization of tyrosinolated alpha-tubulin within individual microtubules and organelles. This localization is subject to a temporal modulation that correlates strongly with progress of a cell through the cell cycle. Our results indicate that the presence of tyrosinolated alpha-tubulin is a marker for newly formed microtubules.

Animals↗
PubMed1987

Antibodies to tubulin in patients with parasitic infections.

Sera from a total of 268 patients with protozoan, helminth, bacterial (leprosy and tuberculosis) infections or appropriate controls, were assayed for anti-tubulin antibodies in an indirect enzyme-linked immunosorbent assay (ELISA), using purified tubulin as antigen. Levels of serum anti-tubulin antibody were significantly elevated in 67% of patients with visceral leishmaniasis, in 60% of patients with cutaneous leishmaniasis, in 89% of patients with onchocerciasis, in 100% of patients with schistosomiasis, and in 94% of patients with leprosy. Little or no increase in anti-tubulin antibody levels was seen in sera from patients with malaria (Plasmodium vivax) or tuberculosis.

Autoantibodies↗
PubMed1986

Amino acid sequence data of alpha-tubulin from myxamoebae of Physarum polycephalum.

About 96% of the amino acid sequence of an alpha-tubulin from the slime mould Physarum polycephalum has been determined. Of 430 sequenced amino acids, 30 differ from the deduced amino acid sequence of a recently published alpha-tubulin complementary DNA from the plasmodial form of P. polycephalum. The myxamoebal alpha-tubulin differs from all other known alpha-tubulins in one of the last three C-terminal amino acids that are Gly-Glu-Tyr instead of the usual Glu-Glu-Tyr. These last three amino acids are preceded by 11 residues that appear to be particularly susceptible to mutation. No heterogeneity was found whilst sequencing the myxamoebal alpha-tubulin, indicating that only one type of alpha-tubulin is present in myxamoebae. This alpha-tubulin appears to be less conserved than the previously described plasmodial alpha-tubulin, supporting the hypothesis that the structural constraints on tubulin in axonemes have a significant effect on its rate of mutation.

Amino Acid Sequence↗
PubMed1986

Amino-acid sequence data of beta-tubulin from Physarum polycephalum myxamoebae.

Starting with 7.7 mg of a beta-tubulin isolated from myxamoebae of the slime mould Physarum polycephalum, 90% of the sequence has been determined by the Edman degradation of peptides generated by cyanogen bromide, trypsin and Staphylococcus aureus protease. Differences to other beta-tubulins are mainly conservative and spread evenly throughout the chain except for a high concentration at the C-terminus. The Physarum beta-tubulin shows most homology to Chlamydomonas beta-tubulin (90.5%) and least homology to yeast beta-tubulin (S. cerevisiae, 73.4%). Two tryptic peptides were isolated in approximately equal quantities which were identical except in one position (S/ALTVPELTQRMFDA) showing that at least two beta-tubulins are present in myxamoebae. However, since this was the only heterogeneity found, these beta-tubulins are probably very similar.

Amino Acid Sequence↗
PubMed1986

Tubulin isotypes: generation of diversity in cells and microtubular organelles.

Diversity of tubulin isotypes is illustrated by consideration of the beta-tubulin isotypes of higher plants and the eukaryotic microbe, Physarum polycephalum, and by the alpha-tubulin isotypes of the protozoan, Trypanosoma brucei. The carrot plant expresses six, well-defined beta-tubulin isotypes that possess characteristic two-dimensional gel coordinates. These six beta-tubulin isotypes are differentially expressed during development of the flowering plant. In a similar manner, Physarum expresses three separate beta-tubuli isotypes during its life cycle; of the two beta 1 isotypes, one is expressed solely in the myxamoeba whilst the other is expressed both in the myxamoeba and in the plasmodium. A further beta-tubulin isotype, beta 2, is expressed only in the plasmodium. In carrot and in Physarum the generation of beta-tubulin diversity appears, in the main, to be generated by the differential expression of a beta-tubulin multi-gene family. However, tubulin isotypes can also be generated by post-translational modifications and T. brucei utilizes two different modifications within one cell. First, the primary translation product, the alpha 1-tubulin isotype, can be acetylated to produce the alpha 3 isotype. Second, both the alpha 1 and alpha 3 isotypes appear to exist in both tyrosinated and detyrosinated forms. The generation of these alpha-tubulin isotypes within the same cell and their presence in particular cellular domains, modulated throughout the cell cycle, reveals a complex relationship between alpha-tubulin isotypes produced by post-translational modifications and the dynamics of microtubule construction.

Animals↗
PubMed1985

Use of monoclonal antibodies to analyse the expression of a multi-tubulin family.

We have used a panel of monoclonal antibodies in a study of the expression of multiple tubulins in Physarum polycephalum. Three anti-beta-tubulin monoclonal antibodies, DM1B, DM3B3 and KMX-1 all reacted with the beta 1-tubulin isotypes expressed in both myxamoebae and plasmodia. However, these antibodies showed a spectrum of reduced reactivity with the plasmodial beta 2-tubulin isotype - the competence of recognition of this isotype was graded DM1B greater than KMX-1 greater than DM3B3. The anti-alpha-tubulin monoclonal antibody, YOL 1/34 defined the full complement of Physarum alpha-tubulin isotypes, whilst the anti-alpha-tubulin monoclonal antibody, KMP-1 showed a remarkably high degree of isotype specificity. KMP-1 recognises all of the myxamoebal alpha 1-tubulin isotypes but only recognises 3 out of the 4 alpha 1-tubulin isotypes expressed in the plasmodium (which normally focus in the same 2D gel spot). KMP-1 does not recognise the plasmodial specific alpha 2-tubulin isotype. This monoclonal antibody reveals a new level of complexity amongst the tubulin isotypes expressed in Physarum and suggests that monoclonal antibodies are valuable probes for individual members of multi-tubulin families.

Antibodies, Monoclonal↗
PubMed1985

Mechanism of action of nikkomycin and the peptide transport system of Candida albicans.

Nikkomycin was found to be a potent growth inhibitor of Candida albicans through competitive inhibition of chitin synthase [Ki = 0.16 microM (0.1 microgram ml-1)]. The activity of the peptide-nucleoside drug was antagonized by both peptone and defined peptides. Transported dipeptides were effective antagonists while transported oligopeptides were not. A mutant of C. albicans resistant to the effects of nikkomycin through a transport defect was unable to transport dipeptides, while oligopeptide uptake was apparently unaffected. At least two peptide permeases are operational in this organism.

Aminoglycosides↗
PubMed1984

A comparison of the interaction of anthelmintic benzimidazoles with tubulin isolated from mammalian tissue and the parasitic nematode Ascaridia galli.

Colchicine and a range of anthelmintic benzimidazoles inhibited the in vitro polymerization of tubulin purified from the parasitic nematode Ascaridia galli. In most cases, this inhibition was more pronounced than that detected when these drugs were incubated with tubulin purified from mammalian tissue. In particular, oxfendazole and thiabendazole had virtually no effect on mammalian tubulin assembly whereas they were both good inhibitors of nematode tubulin polymerization. Electron microscopic examinations revealed no morphological differences between microtubules from either nematode or mammalian tissues polymerized in the presence or absence of drug, though the length and number of microtubules was reduced in the drug-incubated samples. These results show that the benzimidazole group of anthelmintics interacts specifically with nematode tubulin and that their selectivity, at least in part, is a direct consequence of such interaction.

Animals↗
PubMed1984

Cell cycle regulation of tubulin RNA level, tubulin protein synthesis, and assembly of microtubules in Physarum.

The temporal relationship between tubulin expression and the assembly of the mitotic spindle microtubules has been investigated during the naturally synchronous cell cycle of the Physarum plasmodium. The cell cycle behavior of the tubulin isoforms was examined by two-dimensional gel electrophoresis of proteins labeled in vivo and by translation of RNA in vitro. alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin synthesis increases coordinately until metaphase, and then falls, with beta 2 falling more rapidly than beta 1. Nucleic acid hybridization demonstrated that alpha- and beta-tubulin RNAs accumulate coordinately during G2, peaking at metaphase. Quantitative analysis demonstrated that alpha-tubulin RNA increases with apparent exponential kinetics, peaking with an increase over the basal level of greater than 40-fold. After metaphase, tubulin RNA levels fall exponentially, with a short half-life (19 min). Electron microscopic analysis of the plasmodium showed that the accumulation of tubulin RNA begins long before the polymerization of mitotic spindle microtubules. By contrast, the decay of tubulin RNA after metaphase coincides with the depolymerization of the spindle microtubules.

Actins↗
PubMed1984

Genetic analysis of resistance to benzimidazoles in Physarum: differential expression of beta-tubulin genes.

Physarum displays two vegetative cell types, uninucleate myxamoebae and multinucleate plasmodia. Mutant myxamoebae of Physarum resistant to the antitubulin drug methylbenzimidazole-2-yl-carbamate (MBC) were isolated. All mutants tested were cross-resistant to other benzimidazoles but not to cycloheximide or emetine. Genetic analysis showed that mutation to MBC resistance can occur at any one of four unlinked loci, benA, benB, benC or benD. MBC resistance of benB and benD mutants was expressed in plasmodia, but benA and benC mutant plasmodia were MBC sensitive, suggesting that benA and benC encode myxamoeba-specific products. Myxamoebae carrying the recessive benD210 mutation express a beta-tubulin with novel electrophoretic mobility, in addition to a beta-tubulin with wild-type mobility. This and other evidence indicates that benD is a structural gene for beta-tubulin, and that at least two beta-tubulin genes are expressed in myxamoebae. Comparisons of the beta-tubulins of wild-type and benD210 strains by gel electrophoresis revealed that, of the three (or more) beta-tubulin genes expressed in Physarum, one, benD, is expressed in both myxamoebae and plasmodia, one is expressed specifically in myxamoebae and one is expressed specifically in plasmodia. However, mutation in only one gene, benD, is sufficient to confer MBC resistance on both myxamoebae and plasmodia.

Benzimidazoles↗
PubMed1984

Tubulin heterogeneity in the trypanosome Crithidia fasciculata.

The interphase cell of Crithidia fasciculata has three discrete tubulin populations: the subpellicular microtubules, the axonemal microtubules, and the nonpolymerized cytoplasmic pool protein. These three tubulin populations were independently and selectively purified, yielding, in each case, microtubule protein capable of self-assembly. All three preparations polymerized to form ribbons and sheets rather than the more usual microtubular structures. Analyses of the tubulin by two-dimensional polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping indicated that the beta-tubulin complex remained constant regardless of source but that some heterogeneity was present in the alpha subunit. Cytoplasmic pool alpha tubulins (alpha 1/alpha 2) were the only alpha isotypes in the cytoplasm and also formed most of the alpha tubulin species in the pellicular fraction. Flagellar alpha tubulin (alpha 3) was the sole alpha isotype in the flagella; it appeared in small amounts in the pellicular fraction but was completely absent from the cytoplasm. In vitro translation products from polyadenylated RNA from C. fasciculata were also examined by two-dimensional polyacrylamide gel electrophoresis and possessed a protein corresponding to alpha 1/alpha 2 tubulin but lacked any alpha 3 tubulin. The alpha 3 polypeptide arose from a post-translational modification of a precursor polypeptide not identifiable by two-dimensional polyacrylamide gel electrophoresis as alpha 3. Peptide mapping data indicated that cytoplasmic alpha tubulin is the most likely precursor. These results demonstrate alpha-tubulin heterogeneity in this organism and also how close the relationship between flagellar and cytoskeletal tubulins can be among lower eucaryotes.

Animals↗
PubMed1984

Flagellar regeneration of the trypanosome Crithidia fasciculata involves post-translational modification of cytoplasmic alpha tubulin.

Deflagellation of Crithidia fasciculata stimulated formation of new flagella and maximized production of alpha 3 tubulin. Continuous labeling during reflagellation revealed that alpha 1, 2, and 3 tubulins were formed, whereas the polyadenylated RNA translation products lacked alpha 3 isoform. Pulse-chase labeling experiments demonstrated that alpha 3 was a post-translational modification of cytoplasmic alpha tubulin.

Animals↗
PubMed1984

Identification of tubulin isoforms in the plasmodium of Physarum polycephalum by in vitro microtubule assembly.

The tubulins of the plasmodium of Physarum polycephalum have been identified by in vitro microtubule assembly from partially purified extracts of asynchronous microplasmodia and late G2 macroplasmodia. The plasmodial tubulin group comprised of 2 alpha tubulins (app. m.w. 51000 daltons) and 2 beta tubulins (app. m.w. 58000 daltons and 55000 daltons) and appeared to be identical with a group of polypeptides which are synthesized periodically in late G2. Two of the plasmodial tubulin subunits (one alpha and one beta) were identical to the Physarum amoebal tubulin alpha and beta subunits as characterised by 2D gel positions.

Electrophoresis, Polyacrylamide Gel↗
PubMed1984

The isolation of nuclei containing mitotic spindles from the plasmodium of the slime mould Physarum polycephalum.

We have developed a protocol which facilitates the isolation of nuclei containing mitotic spindles from the plasmodium of Physarum polycephalum. The nuclei are isolated in a spindle stabilization buffer (SSB) consisting of -4 M glycerol, 5 mM EGTA, 10 mM MgCl2, 10 mM PIPES and 0.1% (v/v) Triton X-100 at pH 6.5. Isolation at 20 degrees C, chelation of Ca2+ and the inclusion of the 4 M glycerol were found to be essential for stabilization of the spindle microtubules. The method allows the isolation of clean nuclei in a reasonable yield. Nuclei were isolated at each of the main stages of mitosis (prophase, metaphase and anaphase) and transmission electron microscopy of the isolated nuclei revealed well preserved arrangements of spindle microtubules which were characteristic of the particular stage of mitosis.

Cell Nucleus↗