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K Gull

Publications and source records attributed to K Gull.

At least 91 records · Page 5Linked to original sources
PubMed1990

Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei.

We have used immunofluorescent detection of 5-bromo-2-deoxyuridine-substituted DNA in order to determine the timing of initiation and the duration of nuclear and kinetoplast S-phases within the procyclic stage of the Trypanosoma brucei cell cycle. Both nuclear and kinetoplast S-phases were shown to be periodic, occupying 0.18 and 0.12 of the unit cell cycle, respectively. In addition, initiation of both of these S-phases were in approximate synchrony, differing by only 0.03 of the unit cell cycle. We have also used a monoclonal antibody that recognises the basal bodies of T. brucei in order to visualise cells possessing a new pro-basal body and hence determine the time of pro-basal body formation within the cell cycle. Pro-basal body formation occurred within a few minutes of the initiation of nuclear S-phase, at 0.41 of the unit cell cycle. This provides detection of the earliest known cell cycle event in T. brucei at the level of the light microscope. Cell cycle events including initiation of nuclear and kinetoplast DNA replication and pro-basal body formation may be strictly coordinated in T. brucei in order to maintain the precise single-mitochondrion (kinetoplast), singleflagellum status of the interphase cell.

Animals↗
PubMed1989

Recognition of specific Physarum alpha-tubulin isotypes by a monoclonal antibody. Sequence heterogeneity around the acetylation site at lysine 40.

The monoclonal antibody 6-11B-1 recognises specifically the acetylated form of alpha-tubulin. The acetylation event occurs on a unique lysine residue, lysine 40. Using 6-11B-1, acetylated alpha-tubulin was detected in myxamoebae but not plasmodia of Physarum polycephalum. Following chemical acetylation plasmodial alpha-tubulin was detected by 6-11B-1. The monoclonal antibody KMP-1 recognises certain Physarum alpha-tubulin isotypes but only in non-acetylated form. Whilst recognising all the non-acetylated fraction of myxamoebal alpha-tubulin only a proportion of plasmodial alpha-tubulin was recognised by KMP-1. Peptides were synthesised corresponding to the acetylation domains (containing lysine 40) of myxamoebal alpha-tubulin and the inferred acetylation domains of two plasmodial-specific alpha-tubulin isotypes. The only difference between the two peptides was at a single residue corresponding to amino acid 44 in the polypeptide. Tyrosine was present in myxamoebal alpha-tubulin and glycine was present in the plasmodial specific peptides; the peptides are referred to as the Tyr44 and Gly44 peptides respectively. Both peptides in acetylated form blocked 6-11B-1 reactivity towards acetylated myxamoebal alpha-tubulin. The Tyr44 but not the Gly44 peptide blocked KMP-1 reactivity towards non-acetylated myxamoebal alpha-tubulin. Tyrosine at position 44 is not found in any other known alpha-tubulin. Thus a unique antigenic determinant exists in certain Physarum alpha-tubulin isotypes, close to the acetylation site at lysine 40. This antigenic determinant forms part of the KMP-1 recognition epitope and explains the unique isotype selectivity of this monoclonal antibody.

Acetylation↗
PubMed1989

Structure and expression of an alpha-tubulin gene of Physarum polycephalum.

Fragments of Physarum polycephalum DNA generated by partial digestion with Sau3A were cloned into phage-lambda EMBL4. A recombinant (phage-lambda E alpha Tu) containing an alpha-tubulin (E alpha-tubulin) gene was isolated. The E alpha-tubulin gene is part of the alt B locus. The gene was sequenced and was found to contain seven intervening sequences. The alpha-tubulin isotype (E alpha-tubulin) encoded by the gene has a methionine residue at the C-terminus. The E alpha-tubulin gene has much in common with the N alpha-tubulin gene cloned into phage-lambda NM1149 [M. J. Monteiro & R. A. Cox (1987) J. Mol. Biol. 193, 427-438]. However, the gene products E alpha-tubulin and N alpha-tubulin differ in amino acid sequence near to the C-terminus. E-peptide (corresponding to amino acids 440-448 of E alpha-tubulin) and N-peptide (corresponding to amino acids 437-445 of N alpha-tubulin) were synthesised and used to raise antibodies (E-antibodies and N-antibodies). The antibodies were used to show that on two-dimensional gel electrophoresis N alpha-tubulin travels to the alpha 1 position and E alpha-tubulin moves to the alpha 2 position. Gene-specific DNA probes were used to show that transcripts of the E alpha-tubulin gene were present in the plasmodial but not in the amoebal phase of the life cycle. The E- and N-antibodies detected E alpha- and N alpha-tubulins in plasmodia but not in amoebae, confirming that the expression of the E alpha- and N alpha-tubulin genes is regulated during development. E alpha- and N alpha-tubulin were shown to be components of spindle microtubules by indirect immunofluorescence microscopy.

Amino Acid Sequence↗
PubMed1989

Visualization of detyrosination along single microtubules reveals novel mechanisms of assembly during cytoskeletal duplication in trypanosomes.

We have been able to use immunogold labeling with monoclonal antibodies specific for tyrosinated alpha-tubulin to define new microtubule assembly within the T. brucei pellicular cytoskeleton. Using this approach, we have been able to visualize and define the detyrosination gradient along single microtubules in vivo. New microtubules are seen to invade the cytoskeletal array early in the cell cycle between old microtubules. In post-mitotic cells, a unique form of microtubule assembly occurs, with very short microtubules being intercalated in the array. We propose that these are nucleated by lateral interaction with the MAPs on existing adjacent microtubules. This construction pattern suggests a templated morphogenesis of microtubule arrays with semi-conservative distribution to the daughter cells.

Animals↗
PubMed1989

A novel method for the purification of HIV-1 p24 protein from hybrid Ty virus-like particles (Ty-VLPs).

The self-assembly properties of a protein encoded by the yeast retrotransposon Ty can be exploited to produce large amounts of recombinant, particulate fusion proteins as hybrid Ty virus-like particles (Ty-VLPs). This system has now been adapted to allow the release of the additional protein by incorporation of a protease cleavage site between the yeast carrier protein and the protein of interest. The purification of the additional protein is facilitated by exploiting the ease with which Ty-VLPs can be purified from other yeast cell components due to their particulate nature. We have used this modified system to produce hybrid particles containing the HIV-1 p24 protein downstream of the recognition sequence for the blood coagulation factor Xa. The p24 was released from the particles by proteolytic cleavage and rapidly separated from the residual particulate material using centrifugation and standard chromatography techniques. This procedure has been used to purify milligram quantities of HIV-1 p24 protein that reacts with anti-p24 sera and elicits the production of p24-specific antibodies in experimental animals.

Animals↗
PubMed1989

Location of a single beta-tubulin gene product in both cytoskeletal and mitotic-spindle microtubules in Physarum polycephalum.

In the mutant BEN210 of Physarum polycephalum several beta-tubulins are detectable. beta 1-tubulin is unique to the myxamoeba, beta 2-tubulin is unique to the plasmodium, and the mutant beta 1-210 tubulin encoded by the benD210 allele is present in both cell types. In order to analyse the subcellular distribution of the beta 1-210 polypeptide, we prepared cytoskeletons from myxamoebae and mitotic spindles from plasmodia, and examined the tubulin polypeptide composition of these microtubular organelles by two-dimensional gel electrophoresis and immunoblotting. The results show that the beta 1-210 tubulin is present in microtubules of both the cytoskeleton and the intranuclear mitotic spindle. Thus a single beta-tubulin gene product can participate in multiple microtubular organelles in distinct cellular compartments.

Cytoskeleton↗
PubMed1989

Incorporation of tubulin from an evolutionarily diverse source, Physarum polycephalum, into the microtubules of a mammalian cell.

Physarum myxamoebal tubulin was injected into PtK2 cells to determine whether tubulin from this eukaryotic microbe could act as a reporter for microtubule growth and dynamics in a mammalian cell. The distribution of Physarum tubulin was determined by the use of a monoclonal antibody specific for Physarum tubulin and unable to detect mammalian tubulin. Physarum tubulin was incorporated into the microtubules of both interphase arrays and the mitotic spindle. Measurements of microtubule turnover kinetics were found to be similar to those of other studies in which chemically modified brain tubulin has been used. Results using this heterologous system demonstrate that tubulin from an evolutionarily diverse organism can be used as a marker for microtubule growth in mammalian cells. Furthermore, the Physarum tubulin was able to endow the injected cells with novel properties. Resistance to colchicine-induced microtubule disassembly, a characteristic of Physarum tubulin, was conferred on the injected PtK2 cells. Use of this heterologous reporter tubulin system has also revealed features of variation in microtubule dynamics both within individual cells and between cells.

Animals↗
PubMed1989

Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies.

The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.

Animals↗
PubMed1989

Evidence for a Mr 88,000 glycoprotein with a transmembrane association to a unique flagellum attachment region in Trypanosoma brucei.

We have examined the relationship of externally accessible proteins associated with the internal cytoskeleton of procyclic Trypanosoma brucei. Two approaches were taken. First, externally disposed glycoproteins were identified with lectins and examined for their persistence and location in isolated cytoskeletons. Second, proteins containing tyrosine residues available for chemical modification on the outer surface were identified in isolated cytoskeletons and probed for glycosylation. The procyclic form of T. brucei that was employed does not express the variable surface glycoprotein. The lectin concanavalin A (ConA) bound to the outer surface of T. brucei in two discrete locations; one a narrow line close to the flagellum attachment zone on the cell body, the other at the distal tip of the flagellum itself. Of these, only the cell body labelling was detected when isolated cytoskeletons were probed with fluorescein isothiocyanate-labelled ConA. When cytoskeletons were prepared from cells labelled with gold-conjugated ConA, a narrow line of label was detected parallel to the flagellum attachment zone but was distinct from it. Only one cytoskeletal protein, of Mr 88,000, could be labelled at the cell surface by the 125I/iodogen procedure. This protein could be precipitated from SDS-solubilized cytoskeletons with ConA-agarose. These data indicate the existence of a previously undetected cytoskeletal structure, situated in the cell body, close to the point of flagellum attachment, which has a transmembrane association with an external Mr 88,000 glycoprotein.

Animals↗
PubMed1988

Differential and developmental expression of beta-tubulins in a higher plant.

By using two-dimensional gel electrophoresis and immunoblotting, we have analyzed the expression of beta-tubulin isotypes in the higher plant, carrot. We report a complex expression of beta-tubulins that is dependent on the developmental stage of the tissues analyzed. Consequently, each tissue examined can be identified by its unique composition of beta-tubulins. In total, there are six electrophoretically separable beta-tubulins. In no tissue, however, is there less than two or more than five beta-tubulins. Within this framework we have detected a beta-tubulin specific to seedling tissue beta 6, and a beta-tubulin, beta 5, that is found only in the vegetative tissues of the mature plant. Traced from stem to midrib to leaf lamina, the beta 5 isotype becomes progressively dominant relative to beta 1. Another beta-tubulin isotype, beta 4, appears in marked abundance in immature and mature stamens. In isolated mature pollen the beta 4-tubulin overwhelmingly predominates the ubiquitously expressed beta 2-tubulin isotype. The remaining beta-tubulin isotypes also have specific expression programs with beta 1 present in all tissues except pollen and beta 3 absent only from pollen and leafy tissues.

Immunosorbent Techniques↗
PubMed1988

Tubulin post-translational modifications and the construction of microtubular organelles in Trypanosoma brucei.

We have used specific monoclonal antibodies to facilitate a study of acetylated and tyrosinated alpha-tubulin in the microtubule (MT) arrays in the Trypanosoma brucei cell. Acetylated alpha-tubulin is not solely located in the stable microtubular arrays but is present even in the ephemeral microtubules of the mitotic spindle. Moreover, there is a uniform distribution of this isoform in all arrays. Studies of flagella complexes show that acetylation is concomitant with assembly of MTs. There is no subsequent major modulation in the content of acetylated alpha-tubulin in MTs. Conversely, polymerizing flagellar MTs have a high tyrosinated alpha-tubulin content, which is subsequently reduced to a basal level at a discrete point in the cell cycle. The MTs of the intranuclear mitotic spindle appear never to contain tyrosinated alpha-tubulin, suggesting that they are actually constructed of detyrosinated alpha-tubulin or that detyrosination is extremely rapid at this time in the cell cycle. T. brucei therefore, represents a cell type with extremely active mechanisms for the post-translational modification of alpha-tubulin. Our analyses of the timing of acquisition and modulation in relation to MT construction in T. brucei, suggest that acetylation and detyrosination of alpha-tubulin are two independently regulated post-translational modifications, that are not uniquely associated with particular subsets of MTs of defined lability, position or function. Post-assembly detyrosination of alpha-tubulin may provide a mechanism whereby the cell could discriminate between new and old MTs, during construction of the cytoskeleton through the cell cycle. However, we also suggest that continuation of detyrosination, allows the cell, at cell division, to partition into daughter cells two equivalent sets of cytoskeletal MTs.

Acetylation↗
PubMed1988

Variation in cytoplasmic microtubule organization and spindle length between the two forms of the dimorphic fungus Candida albicans.

Candida albicans is a dimorphic fungus capable of growing as a budding yeast and as a filamentous hypha. We have used the technique of immunofluorescence to study the changes in the microtubule cytoskeleton during the cell cycle in both growth forms. This approach has revealed the presence of an extensive system of microtubules, including cytoplasmic microtubules and a rod-like intranuclear spindle. We have provided a complete description of the arrangement of cytoplasmic and spindle microtubules at each phase of the yeast cell cycle. A novel and characteristic feature of the yeast phase of Candida is the presence of an array of short microtubules at the neck of the doublet cell. This neck-associated array (NAA), is apparently organized independently of the main microtubule-organizing centre, the spindle pole bodies, at late anaphase. Analysis of microtubule patterns in the hyphal state reveals that the general arrangements of microtubules are similar to those seen in the yeast phase. These patterns suggest a role for the cytoplasmic microtubules in the nuclear migration that occurs during hyphal growth. A major finding is that the mitotic spindle in hyphae is considerably longer (12-20 microns) than the spindle in yeast cells (7-8 microns). This may reflect the role of the hyphal mitotic spindle not only in nuclear division but also in the positioning of the daughter nuclei at the centres of hyphal compartments.

Candida albicans↗
PubMed1987

The production of hybrid Ty:IFN virus-like particles in yeast.

The yeast retrotransposon Ty encodes proteins that assemble into virus-like particles (Ty-VLPs) which can be readily purified. We have recently shown that expression of the pl protein encoded by the TYA gene of Ty is sufficient for particle formation. In this paper we show that when a heterologous coding sequence, human interferon-alpha 2 (IFN), is fused in frame to the TYA gene, the resulting p1-IFN fusion protein is still assembled into VLPs. These Ty:IFN-VLPs can be easily purified to near homogeneity and furthermore, they induce an antibody response to interferon when they are injected into rabbits. Therefore, these data show that hybrid Ty-VLPs can be used as a convenient system for the efficient purification of fusion proteins in yeast.

Animals↗
PubMed1987

The functions and relationships of Ty-VLP proteins in yeast reflect those of mammalian retroviral proteins.

We have identified the major structural core proteins of Ty virus-like particles (Ty-VLPs) and shown that they are generated by proteolytic cleavage of the primary translation product of TYA, p1. This precursor protein is therefore functionally similar to the gag precursor of retroviruses. Cleavage is mediated by a Ty-encoded protease located at the 5' region of TYB and is accompanied by a change in particle morphology. p1 contains sufficient information for the assembly of a pre-Ty-VLP complex, which does not require the presence of either Ty protease or reverse transcriptase. The results indicate that the requirements and pathway of Ty-VLP formation reflect the initial stages of mammalian retroviral assembly and further support the idea of a common origin for Ty elements and retroviruses.

Amino Acid Sequence↗
PubMed1987

A mutant beta-tubulin confers resistance to the action of benzimidazole-carbamate microtubule inhibitors both in vivo and in vitro.

The mutant BEN210 of Physarum polycephalum is highly resistant to a number of benzimidazole carbamate agents, including methylbenzimidazole-2-yl-carbamate and parbendazole. The resistance is conferred by the benD210 mutation in a structural gene for beta-tubulin. This mutant allele encodes a beta-tubulin with novel electrophoretic mobility. We have used this strain to determine whether the mutant beta-tubulin is used in microtubules and whether this usage permits microtubule polymerisation in the presence of drugs both in vivo and in vitro. In vitro assembly studies of tubulin purified from the mutant strain have shown that microtubules are formed both in the absence of drugs and in all drug concentrations tested (up to 50 microM parbendazole). In contrast, the assembly of microtubules from wild-type tubulin in vitro is totally inhibited by 2-5 microM parbendazole. Thus the resistance of BEN210 to parbendazole observed in vivo has been reproduced in vitro using tubulin purified from the mutant strain. Electrophoretic analysis of the microtubules formed in vitro has shown that both the wild-type and the mutant beta-tubulin are incorporated into the microtubules and that the proportion of mutant to wild-type beta-tubulin appears to remain constant with increasing drug concentration. This is the first demonstration of a single mutation in a tubulin structural gene causing an altered function of the gene product in vitro.

Alleles↗
PubMed1987

Patterns of tubulin isotype synthesis and usage during mitotic spindle morphogenesis in Physarum.

Tubulin synthesis in the naturally synchronous plasmodium of Physarum polycephalum is a markedly periodic event restricted to the late G2 period of the cell cycle. Mitosis in the plasmodium is intranuclear, and there are no cytoplasmic microtubules at any stage of the cell cycle. We have combined a biochemical investigation of the synthesis of the plasmodial tubulin isotypes and their participation in the mitotic spindle with a microscopic study (immunofluorescence) of the development of spindle microtubules throughout the cell cycle. We have shown that all four tubulin isotypes identified in the plasmodium (alpha 1, alpha 2, beta 1 and beta 2) are present in the mitotic spindle. The stoichiometry of isotype usage in the mitotic spindle generally reflects the overall abundance of isotypes in the plasmodium as a whole: beta 2 greater than alpha 1 greater than alpha 2 greater than beta 1. We have also shown that tubulins synthesized in the G2 period of one cell cycle can be incorporated into the spindles of the immediately ensuing mitosis and have sufficient biological longevity to allow participation in the mitotic divisions of future cell cycles. Thus, the phenomenon of periodic tubulin synthesis does not reflect a restricted use of tubulin to the cell cycle in which it was synthesized. The major polymerization of tubulin in the nucleus occurred less than 30 min before metaphase. A novel tubulin-containing structure was, however, present in the nucleus approximately 60 min before metaphase. Polymerized tubulin is rapidly removed from the nucleus following nucleokinesis.

Cell Cycle↗