Α-tubulin Genes Research Articles

Primary structure of an α-tubulin gene of Physarum polycephalum

An α-tubulin gene of Physarum was isolated as a phage-λ NM1149 recombinant (designated phage-λ NαTu). Phage-λ NαTu contained a 4700 base-pair HindIII nuclear DNA fragment of an allele of the altB locus of Physarum (one of four unlinked α-tubulin gene loci). Subfragments of the 4700 base-pair insert of phage-λ NαTu were cloned into phage M13 and the nucleotide sequence was determined by the dideoxy chain termination method. The start point of transcription was identified by primer extension and a putative polyadenylation site was located by S 1 nuclease analysis. The 4650 base-pair HindIII insert into phage-λ NαTu spans the complete gene; sequences upstream from the 5′ end contain the RNA transcription promoter elements (the TATA and CCAAT boxes). The nucleotide sequence encoding α-tubulin contains seven intervening sequences, ranging from 63 to 222 nucleotides in size. The exons have a sequence that is identical with a Physarum α-tubulin cDNA clone, except for three base changes, one leading to a Val codon in place of a Met codon, another leading to a Glu codon in place of an Asp codon, and the third change is silent. The genomic clone provides the nucleotide sequence coding for the last 26 amino acid residues missing from the cDNA clone. The new sequence data indicate that the α-tubulin gene has a C-terminal methionine codon and not a tyrosine codon, which has been found in all α-tubulin genes sequenced to date.

Chromosome location of four genes in Leishmania

Chromosome-sized DNA molecules from Leishmania isolates ( L. mexicana amazonensis, L. mexicana mexicana, L. chagasi, L. major, L. donovani, and L. braziliensis) were separated by orthogonal field alternation gel electrophoresis. The chromosome locations of four genes were mapped. The α-tubulin and rRNA genes each mapped to a single chromosome size class. The β-tubulin and the 5′-spliced-leader-sequence genes were found on more than one chromosome size class and showed variation of hybridization profiles across species.

Genetically essential and nonessential alpha-tubulin genes specify functionally interchangeable proteins.

Microtubules in yeast are essential components of the mitotic and meiotic spindles and are essential for nuclear movement during cell division and mating. The relative importance in these processes of the two divergent alpha-tubulin genes of the budding yeast Saccharomyces cerevisiae, TUB1 and TUB3, was examined through the construction of null mutations and by increasing their copy number on chromosomes and on plasmids. Experiments with null alleles of TUB3 showed that TUB3 was not essential for mitosis, meiosis, or mating. Null alleles of TUB3, however, did cause several phenotypes, including hypersensitivity to the antimicrotubule drug benomyl and poor spore viability. On the other hand, the TUB1 gene was essential for growth of normal haploid cells. Even in diploids heterozygous for a TUB1 null allele, several dominant phenotypes were evident, including slow growth and poor sporulation. This functional difference between the two genes is apparently due to different levels of expression, because extra copies of either gene could suppress the defects caused by a null mutation in the other. We conclude that in spite of the 10% divergence between the products of the two genes, there is no essential qualitative functional difference between them.

Differential expressions of essential and nonessential alpha-tubulin genes in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe has two alpha-tubulin genes and one beta-tubulin gene. Gene disruption experiments showed that the alpha 1-tubulin gene (NDA2) is essential whereas the alpha 2 gene is dispensable. The alpha 2-disrupted cells missing alpha 2 transcript and alpha 2 polypeptide could grow and sporulate normally. The alpha 2 gene, however, was expressed in the wild type and the alpha 1 mutant. Alpha 2-Tubulin was distinguished as an electrophoretic band and was assembled into microtubules. The alpha 2-disrupted cells had an increased sensitivity to an antimicrotubule drug thiabendazole, and the alpha 1(cold-sensitive [cs]) alpha 2 (disrupted) cells became not only cs but also temperature sensitive. Northern blot experiments indicated that alpha 2 transcription was minor and constitutive whereas alpha 1 transcription was major and modulated, depending on the gene copy number of the alpha 2 gene. The amounts of alpha 1 and alpha 2 polypeptides estimated by beta-galactosidase activities of the lacZ-fused genes integrated on the chromosome and by intensities of the electrophoretic bands in crude tubulin fractions, however, were comparable, indicating that alpha 2 tubulin is not a minor subtype. We assume that the cells of Schizosaccharomyces pombe have no excess tubulin pool. alpha 1 mutants would then be blocked in the cell cycle because only half the amount of functional alpha-tubulin required for growth can be produced by the alpha 2 gene. On the other hand, the alpha 2-disrupted cells became viable because the synthesis of alpha 1 tubulin was increased by transcriptional or translational modulation or both. The real cause for essential alpha 1 and dispensable alpha 2 genes seems to be in their regulatory sequences instead of the coding sequences.

Identification of the pleiotropic cell division cycle gene NDA2 as one of two different α-tubulin genes in schizosaccharomyces pombe

Mutations in a cell-cycle gene NDA2 of Schizosaccharomyces pombe have pleiotropic effects on nuclear division, nuclear location, and thiabendazole sensitivity (Toda et al., 1983). By transformation and nucleotide sequence determination, we identified NDA2 as one of two α-tubulin genes present in the genome of S. pombe. Two cloned sequences complemented cold-sensitive and thiabendazole-supersensitive nda2 mutations; one was derived from NDA2 that encodes α1-tubulin, the other from an unidentified locus encoding α2-tubulin. The predicted amino acid sequences showed that the α1 and α2-tubulins had respective residues of 455 and 449 (molecular weights 51,200 and 50,600). The homology to porcine α-tubulin was 76% in both cases. Frequent alterations took place in the two restricted regions. The α1-tubulin ( NDA2) clone had a 90 bp intervening sequence, the α2-tubulin clone did not. RNA blot hybridization experiments indicated that both genes are transcribed. S. pombe tubulin was isolated by cycles of assembly and disassembly. Presumed α- and β-tubulin polypeptide bands reacted with monoclonal antibodies specific for chicken α- and β-tubulins.

Organization and expression of a-tubulin genes in Drosophila melanogaster: One member of the a-tubulin multigene family is transcribed in both oogenesis and later embryonic development

Genomic clones containing α-tubulin sequences were isolated from a library of Drosophila melanogaster DNA and identified by a hybridization-selection and in vitro-translation procedure. The in vitro translation products were identical to the two electrophoretic variants of α-tubulin present in Drosophila embryos. They co-assembled with an embryonic tubulin fraction to form microtubules in vitro and generated the same partial proteolytic fragments as Drosophila α-tubulins. Hybridization in situ to polytene chromosomes revealed that the α-tubulin sequences constitute a multigene family localized on the right arm of chromosome 3 at sites 84 B3–6, 84 D4–8 and 85 E6–10. Clones hybridizing to these sites corresponded to the three major α-tubulin sequences in genomic DNA. The α-tubulin sequences at 84 B3–6 were present twice per haploid genome, embedded in a large duplicated DNA segment. The sequences of the three genomic α-tubulin genes showed considerable divergence, making it possible to conclude that both of the α-tubulin variants present in embryos are encoded by the genes at 84 B3–6. Furthermore, the abundance of this α-tubulin messenger RNA changes with the requirements for microtubule synthesis during embryo development. The genes at 84 B3–6 encoded both the stored maternal mRNA of the oocyte and the major mRNA transcribed during embryonic development.

Isolation of a multigene family containing human α-tubulin sequences

Screening of two human genomic libraries with a chicken α-tubulin complementary DNA probe has resulted in the isolation of several clones containing a common hybridizing region. Restriction mapping of the cloned fragments reveals that, while the majority of sites are retained in each clone, there are a number of differences, including several within the gene-containing region. These differences cannot be ascribed solely to restriction-site polymorphism, and therefore reflect the existence of a family of closely related α-tubulin genes.

α-tubulin genes of drosophila

Four Drosophila α-tubulin genes have been isolated on recombinant DNA molecules. The identity of two of these genes (Tα1 and Tα2) was established by isolating complementary mRNAs and then examining the in vitro translation products of the mRNAs. The one- and two-dimensional gel patterns and the peptide maps of the in vitro products were indistinguishable from those of embryonic α-tubulin. In turn, the embryonic tubulin was identified by determining its amino-terminal sequence. We identified two other cloned α-tubulin genes (Tα3 and Tα4) by their complementarity to Tα1 and Tα2. Maps of restriction endonuclease sites indicate that the four genes are different. DNA hybridization studies demonstrated, however, that three of them have extensive sequence homology with each other and slight homology with the fourth, Tα4. Hybridization to genomic DNA fragments indicated that the four cloned genes account for all of the different α-tubulin genes of Drosophila melanogaster. Three of them are present only once in the haploid genome; the other, Tα1, is present in either one or two copies. Each of the four genes hybridizes in situ to a different site on the third chromosome (67C4-6, 84B3-C8, 84D5-8 and 85E6-15).

Nuclear movement is β-tubulin-dependent in Aspergillus nidulans

We have examined nuclear transport in Aspergillus nidulans to determine whether microtubles function in the movement of this organelle. Nuclear movement was found to be inhibited in germinating conidia (uninucleate asexual spores) by the microtubule inhibitor benomyl. To show that the benomyl inhibition was due to its effect on microtubules, the test was repeated with mutants which have genetic lesions in β-tubulin which produce resistance to benomyl in one case ( benA15) and super-sensitivity in another ( benA16). Nuclear movement was resistant to benomyl in the strain carrying benA15 and super-sensitive in the strain carrying benAl6. Since altered sensitivity to benomyl in these strains is specifically due to alterations in β-tubulin, these results show that β-tubulin is involved in nuclear movement. To eliminate the possibility that nuclear movement blockage was a secondary consequence of nuclear division blockage, this experiment was repeated with temperature-sensitive nuclear division mutants. At restrictive temperature, nuclear division was blocked in these mutants but nuclear movement was not. In the presence of benomyl, nuclear division and migration were blocked at permissive and restrictive temperatures. Thus nuclear division blockage alone is not sufficient to block nuclear movement. These experiments were corroborated by similar experiments on a temperature-sensitive nuclear movement mutant. Five previously isolated nonallelic temperature-sensitive nuclear movement mutants, nudA-E, were analyzed genetically and found not to be allelic to the benA (β-tubulin) tubA α-tubulin genes.