Α-tubulin Genes Research Articles

TUBA8: A New Tissue-Specific Isoform of α-Tubulin That Is Highly Conserved in Human and Mouse

We have cloned and sequenced a cDNA from a human adult skeletal muscle cDNA library, encoding for a novel isoform of α-tubulin (tuba8) that is preferentially expressed in heart, skeletal muscle, and testis. A genomic DNA sequence from the chromosomal region 22q11 allowed us to determine the complete structure of the TUBA8 gene that mirrors the canonical exon/intron organization of the vertebrate α-tubulin genes. We also cloned and sequenced the cDNA of its murine homologue (MMU-TUBA8). The latter encodes for a protein that differs from its human counterpart in only three amino acids, revealing an extreme rate of conservation that is even extended to both the 3′ and 5′ UTRs of the mRNAs. Sequence comparison of these novel isoforms with other known alpha tubulins shows that tuba8 is the most divergent member of the mammalian α-tubulin family. The sequence peculiarity of the human and murine tuba8 strongly suggests that they might have functional significance and, according to the multi-tubulin hypothesis, that they might play specific functional roles in the cell cytoskeleton.

Gene-specific changes in alpha-tubulin transcript accumulation in developing cotton fibers.

The fibers of cotton (Gossypium hirsutum) are single-cell trichomes that undergo rapid and synchronous elongation. Cortical microtubules provide spatial information necessary for the alignment of cellulose microfibrils that confine and regulate cell elongation. We used gene-specific probes to investigate alpha-tubulin transcript levels in elongating cotton fibers. Two discrete patterns of transcript accumulation were observed. Whereas transcripts of alpha-tubulin genes GhTua2/3 and GhTua4 increased in abundance from 10 to 20 d post anthesis (DPA), GhTua1 and GhTua5 transcripts were abundant only through to 14 DPA, and dropped significantly at 16 DPA with the onset of secondary wall synthesis. This is the first report, to our knowledge, of gene-specific changes in tubulin transcript levels during the development of a terminally differentiated plant cell. The decrease in abundance of GhTua1 and GhTua5 transcripts was correlated with pronounced changes in cell wall structure, suggesting that alpha-tubulin isoforms may be functionally distinct in elongating fiber cells. Although total alpha-tubulin transcript levels were much higher in fiber than several other tissues, including the hypocotyl and pollen, none of the alpha-tubulins was specific to fiber cells.

Cloning and sequencing of an α-tubulin cDNA from Artemia franciscana: evidence for translational regulation of α-tubulin synthesis

The brine shrimp, Artemia franciscana, exhibits a limited number of tubulin isotypes which change little during early postgastrula growth. In order to better understand the synthesis of α-tubulins during Artemia development, a cDNA termed αAT1 was cloned and sequenced. Alignment analyses revealed that the polypeptide encoded by αAT1 is similar to α-tubulins from other species. Hybridization of αAT1 to restriction-digested DNA on Southern blots produced a simple banding pattern, indicating that Artemia have a small number of α-tubulin genes. Probing of Northern blots demonstrated an abundant supply of α-tubulin mRNA in dormant cysts, emerging nauplii and instar I larvae. However, it was not until instar I larvae were produced that the amount of polysomal α-tubulin mRNA increased, suggesting that synthesis of the tubulin corresponding to αAT1 is translationally controlled. This work provides one of the few examples where tubulin synthesis is thought to be translationally regulated. Moreover, when considered in the light of previous analyses, the findings imply that cell differentiation in postgastrula Artemia and the diversification of microtubule function certain to accompany this process occur with little or no change in α-tubulin composition.

Effect of vesicular stomatitis virus matrix protein on transcription directed by host RNA polymerases I, II, and III.

The matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and inhibits host-directed gene expression independently of other viral components. Experiments in this study were carried out to determine the ability of M protein to inhibit transcription directed by each of the three host RNA polymerases (RNA polymerase I [RNAPI], RNAPII, and RNAPIII). The effects of wild-type (wt) VSV, v6 (a VSV mutant isolated from persistently infected cells), and tsO82 viruses on poly(A)+ and poly(A)- RNA synthesis were measured by incorporation of [3H]uridine. v6 and tsO82 viruses, which contain M-gene mutations, had a decreased ability to inhibit synthesis of both poly(A)+ and poly(A)- RNA. Nuclear runoff analysis showed that VSV inhibited transcription of 18S rRNA and alpha-tubulin genes, which was dependent on RNAPI and RNAPII, respectively, but infection with wt virus enhanced transcription of 5S rRNA by RNAPIII. The effect of M protein alone on transcription by RNAPI-, RNAPII-, and RNAPIII-dependent promoters was measured by cotransfection assays. M protein inhibited transcription from RNAPI- and RNAPII-dependent promoters in the absence of other viral gene products. RNAPIII-dependent transcription of the adenovirus VA promoters was also inhibited by M protein. However, as observed during wt VSV infection, M protein enhanced endogenous 5S rRNA transcription, indicating that the inhibition of transcription by RNAPIII was dependent on the nature of the promoter.

Identification of novel temperature-sensitive lethal alleles in essential beta-tubulin and nonessential alpha 2-tubulin genes as fission yeast polarity mutants.

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1-15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two alpha-tubulin genes) and alp12 to nda3 (the single beta-tubulin gene). atb2(+) is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between alpha/beta-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2(+); overexpression of atb2(+) lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.

Isolation and characterization of α‐tubulin genes from Septoria tritici and Rhynchosporium secalis, and comparative analysis of fungal α‐tubulin sequences

The α-tubulin genes from Septoria tritici and Rhynchosporium secalis have been cloned and sequenced. The predicted amino acid sequence and intron structure showed strong homology with other known filamentous fungal α-tubulins. Comparison of sixteen fungal α-tubulin sequences based on amino acid sequence homology and intron structure identified five groups of proteins. Group 1 consists of filamentous fungi, including S. tritici and R. secalis, the dimorphic fungus Histoplasma capsulatum, and Pneumocystis carinii. Group 2 includes two divergent isoforms from Neurospora crassa and Aspergillus nidulans. Group 3 includes the yeast Saccharomyces cerevisiae and the dimorphic fungus Candida albicans. Group 4 contains the single yeast Schizosaccharomyces pombe. Group 5 includes the only Basidiomycete, Schizophyllum commune. This analysis supports the classification of P. carinii as a primitive Ascomycete. The presence of an additional glycine residue between the second and third amino acid found only in Group 2 proteins may indicate a functionally distinct fungal isotype. Implications in terms of structure-function relationships for α-tubulin molecules are discussed. Cell Motil. Cytoskeleton 41:247–253, 1998. © 1998 Wiley-Liss, Inc.

Molecular characterization of four members of the α‐tubulin gene family of the Bermuda land crab Gecarcinus lateralis

Four α-tubulin isoforms recovered from a cDNA library from regenerating limb buds of the Bermuda land crab Gecarcinus lateralis have been characterized. Two clones (α1 and α2) contained complete coding sequences with start and stop codons; the other two clones were partial, lacking 5′ ends. The four isoforms showed high homology in their coding sequences but rather low homology in their non-coding regions. Identity between the nucleotide sequences of α1 and α2 was 83.4%; between their predicted amino acid sequences it was 88.9%. The inferred number of amino acid residues for both α1 and α2 was 451, and their calculated molecular weights were 58.29 and 58.45 kDa, respectively. The greatest divergence in the predicted crab α-tubulin proteins occurred near the carboxy terminus, as in α-tubulins of other organisms. When compared with other species, nucleotide sequences of all four clones showed highest homology to α-tubulin genes of an insect (Drosophila melanogaster), while their predicted amino acid sequences were most highly homologous to an α-tubulin of a mammal (Rattus norvegicus). Southern blots revealed a total of five to seven α-tubulin genes encoded in the G. lateralis genome. Northern blots showed single bands of approximately 2.2 kb with an α1-tubulin probe and 1.9 kb with an α2-tubulin probe. mRNA levels of both tubulin isoforms appeared to be independently regulated at different stages of the intermolt cycle in both epidermis and limb buds. Western blots of ID gels of proteins from epidermis, limb buds, or claw muscle showed tissue- and stage-specific changes in tubulin content; similar analyses on blots of 2D gels revealed differences in the number of α-tubulin isoforms that were expressed. J. Exp. Zool. 278:63–77, 1997. © 1997 Wiley-Liss, Inc. 1 This article is a US Government work and, as such, is in the public domain in the United States of America.

Molecular cloning and expression studies of two divergent α-tubulin genes in Neurospora crassa

Three α-tubulin isoforms were previously detected in Neurospora crassa. We have cloned and analysed two α-tubulin cDNAs, Tub αA and Tub αB that encode polypeptides of 453 and 451 amino acids, respectively. The encoded amino acids exhibit an unusual divergence of 35%. This is the highest divergence ever observed between α-tubulins from the same species. The expression of the two genes is developmentally regulated. We did not detect any transcription of the Tub αA gene in dormant macroconidia and during the first 30 min of development even though the α-tub A protein is already present in the early stage of germination. In contrast, the Tub αB gene is continuously transcribed during the vegetative cycle and the expression profile of the protein follows the ones of its mRNA.

Germ-line knockout heterokaryons of an essential alpha-tubulin gene enable high-frequency gene replacement and a test of gene transfer from somatic to germ-line nuclei in Tetrahymena thermophila.

The haploid Tetrahymena thermophila genome contains a single alpha-tubulin (ATU) gene. Using biolistic transformation, we disrupted one of the two copies of the ATU gene in the diploid germ-line micronucleus. The heterozygous germ-line transformants were made homozygous in the micronucleus by mating to a star strain containing a defective micronucleus. This mating, known as round 1 genomic exclusion, resulted in two heterokaryon clones of different mating types which have both copies of the ATU gene knocked out in the micronucleus but only wild-type genes in the polycopy somatic macronucleus. When these heterokaryons were mated, the exconjugant progeny cells did not grow because the new somatic macronuclei do not have any alpha-tubulin genes. However, when these conjugants were transformed with a functional marked ATU gene, viable transformants were obtained that contained the transforming ATU gene at the homologous locus in the new macronucleus. The exconjugant progeny could be rescued at a high efficiency (900 transformants per microg of DNA) with a wild-type ATU gene. Unlike previous macronuclear transformation protocols, this strategy should allow introduction of highly disadvantageous (but viable) mutations into Tetrahymena, providing a powerful tool for molecular and functional studies of essential genes. These knockout heterokaryons were used to demonstrate that gene transfer from somatic macronuclei to germ-line micronuclei occurs rarely if at all.

Endosperm-specific demethylation and activation of specific alleles of alpha-tubulin genes of Zea mays L.

We have investigated the methylation status of the alpha-tubulin genes, and the degree of accumulation of their mRNAs in endosperm, embryo and seedling tissues of Zea mays L. We have found that many of the alpha-tubulin genes are differentially demethylated in the endosperm relative to the embryo and seedling. However, only for tub alpha 2 and tub alpha 4 could a correlation between DNA demethylation and increased RNA accumulation be detected. By analyzing the inbred lines W64A and A69Y and their reciprocal crosses, we have also identified in the endosperm two alpha-tubulin genes, tub alpha 3 and tub alpha 4, that are differentially demethylated if transmitted by the maternal germline, but that remain hypermethylated when transmitted by the paternal germline.

Molecular Characterization of the Tubulin-Related Gene Families in Herbicide Resistant and Susceptible Goosegrass (Eleusine indica)

Goosegrass, wide spread throughout the tropics and subtropics, is one of the most noxious weeds known. Recently, biotypes of goosegrass have been found resistant to the dinitroaniline herbicides. An alteration in the structure/composition of a tubulin protein has been postulated as an explanation for the hyperstable microtubules and the resistant phenotype. Our study was initiated to investigate the structure of the alpha (α)-, beta (β)- and gamma (γ)-tubulin related gene sequences in resistant, intermediately resistant, and susceptible biotypes. Heterologous tubulin gene clones were used as probes of restriction endonuclease-digested genomic DNA from each biotype, to determine gene size and copy number and to screen for restriction fragment length polymorphisms. The tubulin genes are organized into gene families. There are three to five α-tubulin genes, four to seven β-tubulin genes, and four to eight γ-tubulin genes. There was no evidence of multiple copies or tandem repeats of any individual gene sequence. Although RFLPs were observed, no significant difference in the banding pattern between the resistant and the susceptible biotypes was found for either α-, β-, or γ-tubulin gene families. Therefore, it is unlikely that the difference between the herbicide-response phenotypes can be attributed to large deletions or insertions in a tubulin gene.

Estimation of the Number of α-Tubulin Genes in Three Ciliated Protozoa

Summary: In an attempt to study the multitubulin family in ciliated protozoa, we have estimated the number of α-tubulin genes present in the genome of three “Spirotrich” ciliates by using an α-tubulin probe from the ciliate Stylonychia lemnae . Southern hybridization analyses of single or double-digested DNA from ciliates Histriculus cavicola, Euplotes eurystomus and Stentor coeruleus revealed banding patterns consistent with the presence of three, two and at least nine α-tubulin genes in each species, respectively. Comparison of the hybridizing patterns of Histriculus and Euplotes digested DNAs showed a great similarity between both species. The existence of common restriction fragments is an evidence of a sequence homology at the genomic level and might attribute much weight to a close relation between both ciliates. The controversial relationships among “Spirotrich” ciliates are discussed.

A mutation in the α1-tubulin gene of Chlamydomonas reinhardtii confers resistance to anti-microtubule herbicides

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii was isolated by using the amiprophos-methyl-resistant mutation apm1-18 as a background to select new mutants that showed increased resistance to the drug. The upA12 mutation caused twofold resistance to amiprophos-methyl and oryzalin, and twofold hypersensitivity to the microtubule-stabilizing drug taxol, suggesting that the mutation enhanced microtubule stability. The resistance mutation was semi-dominant and mapped to the same interval on linkage group III as the alpha 1-tubulin gene. Two-dimensional gel immunoblots of proteins in the mutant cells revealed two electrophoretically altered alpha-tubulin isoforms, one of which was acetylated and incorporated into microtubules in the axoneme. The mutant isoforms co-segregated with the drug-resistance phenotypes when mutant upA12 was backcrossed to wild-type cells. Two-dimensional gel analysis of in vitro translation products showed that the non-acetylated variant alpha-tubulin was a primary gene product. DNA sequence analysis of the alpha 1-tubulin genes from mutant and wild-type cells revealed a single missense mutation, which predicted a change in codon 24 from tyrosine in wild type to histidine in mutant upA12. This alteration in the predicted amino acid sequence corroborated the approximately +1 basic charge shift observed for the variant alpha-tubulins. The mutant allele of the alpha 1-tubulin gene was designated tua1-1.

α-Tubulin II is a male-specific protein in Plasmodium falciparum

The tubulin gene family in Plasmodium falciparum consists of one β-tubulin and two α-tubulin genes (α-tubulin I and II). We present here data indicating that α-tubulin II is expressed only in male sexual stage parasites. An IgM mAb, 5E7, specifically reacted with stage III (day 4–5) through mature (day 10–11) male gametocytes and with emerging, exflagellating, or freely moving male gametes. No reactivity was detected in female gametocytes, female gametes, sporozoites, or asexual parasites, mAb 5E7 also specifically recognized male gametes of the avian parasite, Plasmodium gallinaceum, and immunoblotted a 50 kDa protein in extracts of male gametes from both species. This 50 kDa antigen was localized by immunoelectron microscopy to axonemes of male gametes in a pattern similar to that obtained with anti-α- and anti-β-tubulin antibodies. Furthermore, mAb 5E7 specifically reacted with recombinant α-tubulin II protein obtained using the PCR-amplified α-tubulin II gene from a gametocyte-specific cDNA library. The sex-specific expression of α-tubulin II and its localization to axoneme of the male parasite suggest a role for this molecule in the morphologic changes that occur during exflagellation and in the motility of the parasite, α-tubulin II and mAb 5E7 may prove useful tools in studies of the biology of sexual stage differentiation and development in P. falciparum in addition to the general understanding of post-translational modifications of tubulin isoforms.

Behavior of structurally divergent α-tubulin isotypes during Drosophila embryogenesis: Evidence for post-translational regulation of isotype abundance

Two major α-tubulin isotypes are present during Drosophila embryogenesis: an evolutionarily divergent maternal isotype that is synthesized only in the ovary and deposited in the oocyte and a highly conserved constitutive isotype that is both maternally supplied and zygotically synthesized. A maternal isotype-specific antibody and a monoclonal antibody that recognizes both the maternal and constitutive isotypes were characterized and used to determine the distribution and abundance of α-tubulins during embryogenesis. Both isotypes are abundant and assemble into all classes of microtubules from the syncytial blastoderm stage until completion of germ band retraction. During subsequent development, however, the maternal isotype is retained only in the developing CNS, and later in a subset of connective fibers within the CNS. In contrast, total α-bubulin levels remain high in essentially all tissues throughout embryogenesis, indicating that most tissues selectively accumulate the constitutive isotype. To determine if selective accumulation of the constitutive isotype requires zygotic synthesis of this protein, mutant embryos that do not contain functional constitutive α-tubulin genes were examined. In these embryos, as in wild type, the maternal isotype decreases to background levels in tissues that retain high levels of the constitutive isotype. The constitutive isotype therefore appears to be more stable than the maternal isotype in most tissues. Differences in isotype stability may play an important role in determining the developmental pattern of isotype accumulation in Drosophila embryos.

Analysis by PCR of the number of homologous genomic sequences to α-tubulin in maize

The protein sequences of α-tubulins are among the best conserved in eukaryotes. This degree of conservation also remains high at the nucleotide level. The genes encoding these proteins are organized into families of up to 30 members in animal systems. In the few plant species studied, the α-tubulin gene family shows a lower complexity. In the reported genomic sequences, for a given species the site of insertion for an intron in the α-tubulin genes occurs at specific points in the gene and this is also true for maize. In this paper, advantage has been taken of the fact that the first intron of maize α-tubulin is variable in length between the different genes reported and it si flanked by coding sequences conserved at both protein and nucleotide levels. These facts offer the possibility of estimating the number of genes coding for this protein in maize. The results and the limitations of the approach are described.

The tubulin genes of the human malaria parasite Plasmodium falciparum, their chromosomal location and sequence analysis of the α-tubulin II gene

We report the isolation and sequencing of genomic clones encompassing the entire α-tubulin II gene from the human malaria parasite Plasmodium falciparum. This gene is closely related to, but significantly different from the α-tubulin I gene that we have described previously [3]. These two genes represent the entire complement of α-tubulin sequences in this organism and are expressed in a stage-specific manner. The α-II gene is present as a single copy and encodes a tubulin molecule with a predicted length of 450 amino acid residues (49.7 kDa). Like the α-I gene, it contains two introns, which are in identical positions to those of α-I, but are about one-third smaller. The deduced α-II protein is very similar to α-tubulin I (94.2% amino acid identity), except for notable differences across residues 40–45. In addition, unlike the great majority of α-tubulin genes (including α-I), α-II does not encode a terminal tyrosine residue. Using pulsed field gel electrophoresis we demonstrate that the two α-tubulin genes, together with the single β-tubulin gene, are unliked, all residing on different chromosomes. We assign α-I to chromosome 9, α-II to chromosome 4 and β-tubulin to chromosome 10.

Functional implications of the unusual spatial distribution of a minor α-tubulin isotype in Drosophila: A common thread among chordotonal ligaments, developing muscle, and testis cyst cells

Three of the four α-tubulin genes in Drosophila melanogaster are temporally regulated. mRNA from one of these genes, α85E-tubulin, first appears in 6- to 8-hr embryos and persists, with marked fluctuations, through the end of pupal development. In adults, α85E mRNA has been unequivocally identified only in testes. In the present study, isotype-specific antibodies have been used to localize α85E tubulin protein in whole tissues. The results demonstrate a spatially restricted expression pattern of the α85E gene that includes tissues of both ectodermal and mesodermal origins. Specifically, embryonic accumulation of α85E tubulin is limited to support cells of chordotonal organs and the developing musculature of the viscera and body wall. In late third instar larvae, chordotonal organs and a subset of larval nerves, but not muscle, stain with anti-α85E. The timing of protein accumulation during pupal development suggests that α85E tubulin is involved in the construction of the adult as well as the larval musculature. In testis, only the somatically derived cyst cells that surround developing spermatid bundles accumulate α85E-tubulin. The cell types that express α85E share a requirement for extensive cell shape changes during development, suggesting that this minor α-tubulin may have distinct functional properties.

Developmental distribution of RNA and protein products of the Drosophila α-tubulin gene family

The developmental pattern of gene expression of the Drosophila melanogaster α-tubulin family has been examined in detail at both the mRNA and protein levels. Northern data from 16 stages of development have been quantified to produce estimates of pool sizes of each of the α-tubulin transcripts through development. The in situ pattern of α67C RNA localization in developing oocytes and early embryos has also been determined. At the protein level, two of the three previously unidentified products of α-tubulin genes (α67C and α85E) have been identified. Evidence that protein from the fourth gene comigrates with the ubiquitously expressed α84B is presented. In addition to the primary translational products of the α-tubulin genes, an elaborate series of post-translationally modified α-tubulins has been resolved. The developmental profiles of both synthesis and accumulation of these α-tubulin proteins are described.

Molecular clones of α-tubulin genes of Plasmodium yoelii reveal an unusual feature of the carboxy terminus

By using a Trypanosoma brucei α-tubulin cDNA probe under reduced stringency hybridization conditions, we have isolated two genomic clones that constitute portions of α-tubulin genes of the rodent malarial parasite Plasmodium yoelii, P. yoelii has two α-tubulin genes, the 3′ portions of which were present in the two clones, PyαT1 and PyαT2, containing 1.3 kb and 6.6 kb EcoRI fragments respectively. The 1358 bp PyαT1 clone was completely sequenced and found to contain 591 nucleotides of uninterrupted coding sequence with a strong bias for AT-rich codons, starting with codon 254 of a consensus α-tubulin sequence. Numerous attempts to clone 5′ portions of these genes were unsuccessful. A single mRNA of 2.3 kb was recognized by both the clones in the erythrocytic stages of P. yoelii. A probe constituting the untranslated sequences of PyαT1 also recognized this RNA but failed to hybridize with PyαT2 sequences, indicating that the gene represented by the PyαT1 clone was expressed during the erythrocytic stages. The deduced amino acid sequence of the PyαT1 gene terminates in Tyr-Glu instead of Glu-Tyr observed in α-tubulins of almost all other organisms. The difference observed may have implications for α-tubulin metabolism in malarial parasites.