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.     

Retention of autoregulatory control of tubulin synthesis in cytoplasts: demonstration of a cytoplasmic mechanism that regulates the level of tubulin expression

Virtually all animal cells rapidly and specifically depress synthesis of new alpha- and beta-tubulin polypeptides in response to microtubule inhibitors that increase the pool of depolymerized subunits, or in response to direct elevation of the cellular tubulin subunit content through microinjection of exogenous tubulin subunits. Collectively, these previous findings have documented the presence of an apparent eucaryotic, autoregulatory control mechanism that specifies the level of expression of tubulin in cultured animal cells. Mechanistically, this regulation of tubulin synthesis is achieved through modulation of tubulin mRNA levels. To dissect further the molecular pathway that underlies this autoregulatory phenomenon, we have now investigated whether enucleated cells still retain the requisite regulatory machinery with which to alter tubulin synthetic levels in response to fluctuations in the pool size of unpolymerized tubulin subunits. Using two-dimensional gel electrophoresis to analyze the patterns of new polypeptide synthesis, we have determined that such cytoplasts can indeed respond to drug-induced microtubule depolymerization by specific repression of new beta-tubulin synthesis. Moreover, the response of cytoplasts is, if anything, greater in magnitude than that of whole cells. We conclude that autoregulatory control of beta-tubulin gene expression must derive principally, if not exclusively, from a cytoplasmic control mechanism that modulates beta-tubulin mRNA stability. For alpha-tubulin, although the response of cytoplasts after drug-induced microtubule depolymerization is quantitatively less dramatic than that of whole cells, at least part of the regulatory machinery must also be activated through a cytoplasmic regulatory event.     

Sequences that confer beta-tubulin autoregulation through modulated mRNA stability reside within exon 1 of a beta-tubulin mRNA

Synthesis of alpha- and beta-tubulin is controlled in animal cells by a novel autoregulatory mechanism: the concentration of unpolymerized subunits specifies the level of tubulin mRNAs. Using transient DNA transfection, we have localized the sequences that identify a beta-tubulin RNA as a substrate for autoregulation. Insertion of as few as 106 nucleotides (57 bases of 5' untranslated region and 49 coding nucleotides) from a beta-tubulin gene into a thymidine kinase gene is sufficient to make expression of the resultant chimeric RNA regulated as if it were an authentic beta-tubulin mRNA. Furthermore, all 5' untranslated region sequences can be deleted without disrupting regulation. We conclude that this novel autoregulatory pathway is specified by cytoplasmic events that modulate mRNA stability through sequences lying within the first 16 translated codons of a beta-tubulin mRNA.     

A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation

Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the alpha- and beta-tubulin subunits. The isotype sequences are conserved across species supporting the hypothesis that different isotypes subserve different functions. To date, however, most studies have demonstrated that tubulin isotypes are freely interchangeable and coassemble into all classes of microtubules. We now report that, in contrast to other isotypes, overexpression of a mouse class V beta-tubulin cDNA in mammalian cells produces a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes also disrupt mitotic spindle assembly and block cell proliferation. Consistent with diminished microtubule assembly, there is an increased tolerance for the microtubule stabilizing drug, paclitaxel, which is able to reverse many of the effects of class V beta-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V beta-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V beta-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates.     

Dinitroaniline herbicide-resistant transgenic tobacco plants generated by co-overexpression of a mutant alpha-tubulin and a beta-tubulin.

Dinitroaniline herbicides are used for the selective control of weeds in arable crops. Dinitroaniline herbicide resistance in the invasive weed goosegrass was previously shown to stem from a spontaneous mutation in an alpha-tubulin gene. We transformed and regenerated tobacco plants with an alpha/beta-tubulin double gene construct containing the mutant alpha-tubulin gene and showed that expression of this construct confers a stably inherited dinitroaniline-resistant phenotype in tobacco. In all transformed lines, the transgene alpha- and beta-tubulins increased the cytoplasmic pool of tubulin approximately 1.5-fold while repressing endogenous alpha- and beta-tubulin synthesis by up to 45% in some tissues. Transgene alpha- and beta-tubulin were overexpressed in every plant tissue analyzed and comprised approximately 66% of the total tubulin in these tissues. Immunolocalization studies revealed that transgene alpha- and beta-tubulins were incorporated into all four microtubule arrays, indicating that they are functional. The majority of the alpha/beta-tubulin pools are encoded by the transgenes, which implies that the mutant alpha-tubulin and the beta-tubulin can perform the majority, if not all, of the roles of microtubules in both juvenile and adult tobacco plants.     

Regulation of tubulin synthesis and cell cycle progression in mammalian cells by gamma-tubulin-mediated microtubule nucleation

We have previously shown that gamma-tubulin, the third member of the tubulin family that functions in microtubule nucleation, when overexpressed, accumulates throughout the cytoplasm and forms numerous ectopic microtubule nucleation sites in mammalian cells (Shu and Joshi [1995] J. Cell. Biol. 130:1137-1147). We now show that overexpression of gamma-tubulin differentially upregulates the synthesis of alpha- and beta-tubulins in mammalian cells. Surprisingly, despite a dramatic increase in the level of gamma-tubulin protein in transfected cells, there is no obvious alteration in the level of endogenous gamma-tubulin mRNA, suggesting that synthesis of gamma-tubulin might employ a regulatory mechanism other than the autoregulatory pathway shared by alpha- and beta-tubulins. Interestingly, a significant number of mammalian cells transfected with gamma-tubulin fail to form normal bipolar mitotic spindle during mitosis; instead, numerous microtubules occur in the cytoplasm intermingled with the condensed chromosomes. In addition, they reduplicate their DNA after an abnormal mitotic exit. These results thus suggest that the number of microtubule nucleation sites, or even gamma-tubulin itself, might play an important role in the regulation of tubulin synthesis as well as cell cycle progression.     

Regulation of tubulin synthesis during the cell cycle in the synchronous plasmodia of Physarum polycephalum

Regulation of alpha- and beta-tubulin isotype synthesis during the cell cycle has been studied in the myxomycete Physarum polycephalum, by subjecting synchronous plasmodia to temperature shifts and pharmacological perturbations. Temperature shifts interfered with the regulation of tubulin synthesis. Inhibition of DNA synthesis prevents tubulin degradation after completion of the cell cycle (Ducommun and Wright, Eur. J. Cell Biol., 50:48-55, 1989) but did not perturb the initiation of tubulin synthesis. The constant increase of tubulin synthesis in the presence of tubulin-sequestering drugs and the decrease of tubulin synthesis during a treatment with aphidicolin in late G2 phase suggest the existence of an autoregulatory mechanism of tubulin synthesis. Moreover, the microtubule poison methyl benzimidazole carbamate dissociated synthesis of the alpha 1-tubulin isotype from the generally strictly coordinated synthesis of all tubulin isotypes during the transient interruption of mitosis. These observations show that a microtubular poison can perturb regulation of the synthesis of specific isotubulins.     

Tubulin expression in trypanosomes

Microtubules in trypanosomes are the main component of the flagellar axoneme and of the subpellicular microtubule corset, whose relative positions determine the morphology of each cell stage of the life cycle of these parasites. Microtubules are polymers of tubulin, a protein dimer of two 55-kDa subunits, alpha- and beta-tubulin; in Trypanosoma brucei, the tubulin-coding sequences are clustered in a 40-kb fragment of tandemly repeated alpha- and beta-tubulin genes separated by a 170-bp intergenic zone. This cluster is transcribed in a unique RNA which is rapidly processed into mature mRNAs carrying the 5' 35-nucleotide leader sequence found in all trypanosome mRNAs. Although no heterogeneity has been found at the gene level, tubulin can be post-translationally modified in 2 ways: the C-terminal tyrosine of alpha-tubulin can be selectively cleaved and added again with 2 enzymes, tubulin carboxypeptidase and tubulin-tyrosine ligase; alpha-tubulin can also be acetylated on a lysine residue. Some molecular domains of tubulin are restricted to subpopulations of microtubules; for instance, the beta-tubulin form defined by the monoclonal antibody 1B41 is sequestered into a part of the subpellicular cytoskeleton limited to the flagellar adhesion zone, which might correspond to the group of 4 microtubules associated with a cisterna of the endoplasmic reticulum, forming the so-called "subpellicular microtubule quartet" (SFMQ). The early assembly of this zone in each daughter cell during the cell division of T. brucei, together with the alterations undergone by the domain defined by the monoclonal antitubulin 24E3 during the differentiation of Trypanosoma cruzi, suggest that specific tubulin forms are responsible for dynamic properties of SFMQ possibly involved in trypanosome morphogenesis.     

Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins.

We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.     

Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.