Transfer and amplification of a mutant beta-tubulin gene results in colcemid dependence: use of the transformant to demonstrate regulation of beta-tubulin subunit levels by protein degradation.

Total genomic DNA from a temperature-sensitive, colcemid-resistant Chinese hamster ovary (CHO) cell mutant expressing an electrophoretic variant beta-tubulin was used to transform wild-type CHO cells to colcemid-resistant cells at 37 degrees C. Southern blot analysis of the transformant demonstrated the three- to fivefold amplification of one of many beta-tubulin sequences compared with that of the wild type or mutant, thereby identifying a functional tubulin gene in CHO cells. This amplification of one tubulin-coding sequence resulted in a threefold increase in two beta-tubulin mRNA species, suggesting that both species may be encoded by a single gene. Pulse-chase experiments showed that in the transformant, total beta-tubulin was synthesized and degraded faster than in the revertant or wild-type cells, so that the steady-state levels of beta-tubulin and alpha-tubulin were unchanged in the transformant compared with those of wild-type, mutant, or revertant cells. Increased ratios of mutant to wild-type beta-tubulin made the transformant dependent on microtubule-depolymerizing drugs for growth at 37 but not 34 degrees C and supersensitive to the microtubule-stabilizing drug taxol at 34 degrees C.     

Cold-adapted tubulins in the glacier ice worm, Mesenchytraeus solifugus

Glacier ice worms, Mesenchytraeus solifugus and related species, are the only known annelids that survive obligately in glacier ice and snow. One fundamental component of cold temperature adaptation is the ability to polymerize tubulin, which typically depolymerizes at low physiological temperatures (e.g., <10 degrees C) in most temperate species. In this study, we isolated two alpha-tubulin (Msalpha1, Msalpha2) and two beta-tubulin (Msbeta1, Msbeta2) subunits from an ice worm cDNA library, and compared their predicted amino acid sequences with homologues from other cold-adapted organisms (e.g., Antarctic fish, ciliate) in an effort to identify species-specific amino acid substitutions that contribute to cold temperature-dependent tubulin polymerization. Our comparisons and predicted protein structures suggest that ice worm-specific amino acid substitutions stabilize lateral contact associations, particularly between beta-tubulin protofilaments, but these substitutions occur at different positions in comparison with other cold-adapted tubulins. The ice worm tubulin gene family appears relatively small, comprising one primary alpha- and one primary beta-tubulin monomers, though minor isoforms and psuedogenes were identified. Our analyses suggest that variation occurs in the strategies (i.e., species-specific amino acid substitutions, gene number) by which cold-adapted taxa have evolved the ability to polymerize tubulin at low physiological temperatures.     

Cloning and expression of the tubulin genes in barley

Tubulins are encoded by small gene families in plants. Based on the barley EST collection, cDNAs for alpha-, beta-, and gamma-tubulins were selected. Five genes for alpha-tubulin, eight newly identified beta-tubulin sequences and one gamma-tubulin gene were found to be expressed in barley. In silico analysis of relative abundance of the distinct tubulin sequences among ESTs derived from different libraries revealed that the various tubulin genes differed in their level of expression, and to some extent were tissue specific.     

Regulation of tubulin levels and microtubule assembly in Saccharomyces cerevisiae: consequences of altered tubulin gene copy number.

Microtubule organization in the cytoplasm is in part a function of the number and length of the assembled polymers. The intracellular concentration of tubulin could specify those parameters. Saccharomyces cerevisiae strains constructed with moderately decreased or increased copy numbers of tubulin genes provide an opportunity to study the cellular response to a steady-state change in tubulin concentration. We found no evidence of a mechanism for adjusting tubulin concentrations upward from a deficit, nor did we find a need for such a mechanism: cells with no more than 50% of the wild-type tubulin level were normal with respect to a series of microtubule-dependent properties. Strains with increased copies of both alpha- and beta-tubulin genes, or of alpha-tubulin genes alone, apparently did down regulate their tubulin levels. As a result, they contained greater than normal concentrations of tubulin but much less than predicted from the increase in gene number. Some of this down regulation occurred at the level of protein. These strains were also phenotypically normal. Cells could contain excess alpha-tubulin protein without detectable consequences, but perturbations resulting in excess beta-tubulin genes may have affected microtubule-dependent functions. All of the observed regulation of levels of tubulin can be explained as a response to toxicity associated with excess tubulin proteins, especially if beta-tubulin is much more toxic than alpha-tubulin.     

Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins.

Two alpha-tubulin genes from the budding yeast Saccharomyces cerevisiae were identified and cloned by cross-species DNA homology. Nucleotide sequencing studies revealed that the two genes, named TUB1 and TUB3, encoded gene products of 447 and 445 amino acids, respectively, that are highly homologous to alpha-tubulins from other species. Comparison of the sequences of the two genes revealed a 19% divergence between the nucleotide sequences and a 10% divergence between the amino acid sequences. Each gene had a single intervening sequence, located at an identical position in codon 9. Cell fractionation studies showed that both gene products were present in yeast microtubules. These two genes, along with the TUB2 beta-tubulin gene, probably encode the entire complement of tubulin in budding yeast cells.     

OP18/stathmin binds near the C-terminus of tubulin and facilitates GTP binding.

It is has been previously suggested that the protein Op18/stathmin may interact with tubulin via the alpha-tubulin subunit [Larsson, N., Marklund, U., Melander Gradin, H., Brattsand, G. & Gullberg, M. (1997) Mol. Cell. Biol. 17, 5530-5539]. In this study we have used limited proteolysis and cross-linking analysis to localize further the stathmin-binding site on alpha-tubulin. Our results indicate that such a binding site is in a region close to the C-terminus of the molecule comprising residues 307 to the subtilisin-cleavage site on the alpha-tubulin subunit. Based on a recent model of the structure of tubulin [Nogales, E., Wolf, S.G. & Dowing, D.H. (1998) Nature (London) 391, 199-203], we found that this region contained the same areas that may be involved in longitudinal contacts of alpha-tubulin subunits within the microtubule. We also observed that the binding of stathmin to tubulin can modulate the binding of GTP to tubulin, as a consequence of a conformational change in the beta-tubulin subunit that occurs upon interaction of stathmin with tubulin.     

Multiple alpha- and beta-tubulin genes in Chlamydomonas and regulation of tubulin mRNA levels after deflagellation

We constructed and characterized recombinant cDNA clones containing alpha- and beta-tubulin DNA sequences. The inserted DNA was determined to code for alpha- and beta-tubulin by positive hybridization-selection. The selected mRNA was translated in vitro, and the translation products were shown to be alpha- or beta-tubulin by comigration with flagellar alpha- and beta-tubulin on one- and two-dimensional gels and by immunoprecipitation with antibodies specific for alpha- and beta-tubulin. Hybridization of the cloned tubulin probes with Chlamydomonas DNA indicated that there are at least two genes each for alpha- and beta-tubulin in this organism. No evidence of cross-hybridization between alpha- and beta-tubulin DNA sequences was found. Because previous experiments had shown that tubulin synthesis was stimulated in response to flagellar amputation, the tubulin clones were used to analyze the levels of tubulin sequences in RNA from cells before and after deflagellation. Hybridization of the tubulin cDNA probes with total or polyadenylated RNA indicated that tubulin sequences in RNA increased within 8 min following deflagellation, reached maximal levels by 50 min and began to decrease by 80 min after deflagellation. One hybridization band was detected with use of the beta-tubulin probe, but RNA in two size classes hybridized to the alpha-tubulin probe.     

Low resolution structure of microtubules in solution. Synchrotron X-ray scattering and electron microscopy of taxol-induced microtubules assembled from purified tubulin in comparison with glycerol and MAP-induced microtubules.

The structure of microtubules has been characterized to 3 nm resolution employing time-resolved X-ray scattering. This has revealed detailed structural features of microtubules not observed before in solution. The polymerization of highly purified tubulin, induced by the antitumour drug taxol, has been employed as a microtubule model system. This assembly reaction requires Mg2+, is optimal at a 1:1 taxol to tubulin heterodimer molar ratio, proceeds with GTP or GDP and is intrinsically reversible. The X-ray scattering profiles are consistent with identical non-globular alpha and beta-tubulin monomers ordered within the known helical surface lattice of microtubules. Purified tubulin-taxol microtubules have a smaller mean diameter (approx. 22 nm) than those induced by microtubule associated proteins or glycerol (approx. 24 nm), but nearly identical wall substructure to the resolution of the measurements. This is because the majority of the former consist of only 12 protofilaments instead of the typical 13 protofilaments, as confirmed by electron microscopy of thin-sectioned, negatively stained and ice-embedded taxol microtubules. It may be concluded that taxol induces a slight reduction of the lateral contact curvature between tubulin monomers. The main fringe pattern observed in cryo-electron micrographs is consistent with a simple 12 protofilament 3-start skewed lattice model. Cylindrical closure of this lattice can be achieved by tilting the lattice 0.8 degrees with respect to the microtubule axis. The closure implies a discontinuity in the type of lateral contacts between the tubulin monomers (regardless of whether these are of the -alpha-beta- or the -alpha-alpha-/-beta-beta- type), which indicates that lateral contacts and the subunit specificity of taxol binding are, to a large degree, equivalent.     

The molecular karyotype of Leishmania major and mapping of alpha and beta tubulin gene families to multiple unlinked chromosomal loci.

The arrangement of tubulin genes in the genome of the protozoan parasite Leishmania major was studied by genomic Southern blot analysis and mapping of genes to chromosomes fractionated by pulsed field gradient gel (PFG) electrophoresis. alpha-tubulin genes exist as a tandem array of 2.4 kb PstI fragments. beta-tubulin genes are found as a tandem array of 3.9 kb AvaI or PvuI fragments, but additional genes are also found on other genomic DNA fragments. Chromosome-sized DNA molecules released from promastigotes of L. major were fractionated into at least 17 chromosome bands of approximate size 400-4000 kb by PFG gel electrophoresis. Some bands may be present in non-equimolar amounts suggesting that there may be more than 17 chromosomes. All alpha-tubulin genes were localized to a single band (chromosome 7). beta-tubulin genes were localized to four bands (chromosomes 6, 10, 16 and 17). This shows that the alpha- and beta- tubulin gene families are unlinked in L. major. There is a single chromosomal locus for the alpha-tubulin tandem array whereas beta-tubulin genes exist both as a tandem array and as dispersed genes at four chromosomal loci.     

Pig sperm tail tubulin. Its extraction and characterization

Axonemal tubulin extracted from pig sperm tails has been characterized by one- and two-dimensional electrophoresis and by one-dimensional peptide mapping. The electrophoretic mobilities of its subunits after reduction and carboxymethylation were similar to those of the major subunits of pig brain tubulin. Sperm tail tubulin subunits also had roughly the same isoelectric points as pig brain tubulin subunits, except that they appeared to have a relatively larger tailing effect. The proteolytic cleavage pattern of the pig sperm tail beta-tubulin closely resembled those of both the tunicate (Ciona intestinalis) sperm beta-tubulin and pig brain beta-tubulin. The peptide pattern of pig sperm tail alpha-tubulin, however, was more similar to that of tunicate sperm tail alpha-tubulin than to that of pig brain alpha-tubulin. This supports the hypothesis put forward in a previous investigation [1] that functionally similar tubulins from taxonomically distant species can be more related than functionally dissimilar tubulins from the same species.     

Taxol-dependent mutants of Chinese hamster ovary cells with alterations in alpha- and beta-tubulin

Chinese hamster ovary cell mutants resistant to the microtubule stabilizing drug taxol were isolated in a single step. Of these 139 drug-resistant mutants, 59 exhibit an absolute requirement for taxol for normal growth and division, 13 have a partial requirement, and 69 grow normally without the drug. Two-dimensional gel analysis of whole cell proteins reveal "extra" spots representing altered tubulins in 13 of the mutants. Six of these have an altered alpha-tubulin and seven have an altered beta-tubulin. Cells with an absolute dependence on taxol become large and multinucleated when deprived of the drug. In contrast, partially dependent cells exhibit some multinucleation, but most cells appear normal. In one mutant that has an absolute dependence on taxol, the cells appear to die more quickly and their nuclei do not increase in size or number. As previously found for another taxol-dependent mutant (Cabral, F., 1983, J. Cell. Biol., 97:22-29), the taxol dependence of the mutants described in this paper behaves recessively in somatic cell hybrids, and the cells are more susceptible to being killed by colcemid than are the wild-type parental cells. When compared with wild-type cells, taxol-dependent mutants have normal arrays of cytoplasmic microtubules but form much smaller mitotic spindles in the presence of taxol. When deprived of the drug, however, these mutants cannot complete assembly of the mitotic spindle apparatus, as judged by tubulin immunofluorescence. Thus, the defects leading to taxol dependence in these mutants with defined alterations in alpha- and beta-tubulin appear to result from the cell's inability to form a functional mitotic spindle. Reversion analysis indicates that the properties of at least one alpha-tubulin mutant are conferred by the altered tubulin seen on two-dimensional gels.