Beta-tubulin Isotype Research Articles

In vivo microtubules are copolymers of available beta-tubulin isotypes: localization of each of six vertebrate beta-tubulin isotypes using polyclonal antibodies elicited by synthetic peptide antigens.

beta-Tubulin is encoded in the genomes of higher animals by a small multigene family comprising approximately seven functional genes. These genes produce a family of closely related, but distinct polypeptide isotypes that are distinguished principally by sequences within the approximately 15 carboxy-terminal amino acid residues. By immunizing rabbits with chemically synthesized peptides corresponding to these variable domain sequences, we have now prepared polyclonal antibodies specific for each of six distinct isotypes. Specificity of each antiserum has been demonstrated unambiguously by antibody binding to bacterially produced, cloned proteins representing each isotype and by the inhibition of such binding by preincubation of each antiserum only with the immunizing peptide and not with heterologous peptides. Protein blotting of known amounts of cloned, isotypically pure polypeptides has permitted accurate quantitative measurement of the amount of each beta-tubulin isotype present in the soluble and polymer forms in various cells, but has not revealed a bias for preferential assembly of any isotype. Localization of each isotype in three different cell types using indirect immunofluorescence has demonstrated that in vivo each class of microtubules distinguishable by light microscopy is assembled as copolymers of all isotypes expressed in a single cell.

The sequence and expression of the divergent beta-tubulin in chicken erythrocytes.

We report here the complete sequence of a highly divergent chicken erythrocyte beta-tubulin, c beta 6, which appears to represent a major exception to the observation that the primary sequences and sites of expression of beta-tubulin isotypes are conserved within vertebrates. The amino acid sequence was deduced from overlapping cloned cDNAs identified in a chicken erythroblast cDNA library contained in the expression vector, lambda gt11. Compared with other chicken beta-tubulins, among which the maximum sequence divergence is only 8%, c beta 6-tubulin is more hydrophobic, contains seven fewer net negative charges, and exhibits a surprising 17% overall divergence in its amino acid sequence. DNA and RNA blot analyses show that c beta 6-tubulin is present as a single gene copy in the chicken genome and is specifically expressed in the bone marrow. Comparisons of RNA blots and immunoblots of various cells and tissues confirm that this beta-tubulin isotype is contained specifically in erythrocytes and thrombocytes and accounts for 75% of the beta-tubulin mRNA species contained in developing erythroblasts. Interestingly, c beta 6-tubulin exhibits 18% amino acid sequence divergence relative to MB1, the analogous hematopoietic beta-tubulin contained in mouse.

Cloning of cDNAs for two beta-tubulin isotypes expressed in murine T cell lymphoma, L5178Y and analysis of their translation products.

Three beta-tubulin isoforms, M beta Ib, M beta Ia and M beta II, were detected by isoelectric focusing gel electrophoresis (IEF) in extracts of cultured cells of a mouse T cell lymphoma, L5178Y. To investigate the origin(s) of the isoforms, we have isolated cDNA clones encoding beta-tubulin from a cDNA library prepared from poly (A)+ RNA of L5178Y cells. Seventeen cDNA clones carrying the entire coding sequences of beta-tubulin were isolated and classified into two distinct isotypes, represented by two clones designated pMT27 and pMT49, according to the results of restriction mapping. On the basis of the nucleotide sequences of the two cDNAs, pMT27 and pMT49 were identified as mouse beta-tubulin isotypes 3 and 5, respectively. By using in vitro translation products of hybrid-selected mRNAs and of the SP6 in vitro transcripts of the cDNAs, polypeptides encoded by the two cDNA clones were analyzed by IEF. We found that pMT27 and pMT49 encode M beta Ib and M beta Ia, respectively. In addition, M beta II was detected in translation products of mRNA specifically hybridized to pMT49, but not in those of the in vitro transcript of pMT49 DNA. These results suggest that M beta II is the translation product of mRNA whose 3'-untranslated region is highly homologous to that of pMT49.

The mammalian beta-tubulin repertoire: hematopoietic expression of a novel, heterologous beta-tubulin isotype.

We describe the structure of a novel and unusually heterologous beta-tubulin isotype (M beta 1) isolated from a mouse bone marrow cDNA library, and a second isotype (M beta 3) isolated from a mouse testis cDNA library. Comparison of M beta 1 and M beta 3 with the completed (M beta 4, M beta 5) or extended (M beta 2) sequence of three previously described beta-tubulin isotypes shows that each includes a distinctive carboxy-terminal region, in addition to multiple amino acid substitutions throughout the polypeptide chain. In every case where a mammalian interspecies comparison can be made, both the carboxy-terminal and internal amino acid substitutions that distinguish one isotype from another are absolutely conserved. We conclude that these characteristic differences are important in determining functional distinctions between different kinds of microtubule. The amino acid homologies between M beta 2, M beta 3, M beta 4, and M beta 5 are in the range of 95-97%; however the homology between M beta 1 and all the other isotypes is very much less (78%). The dramatic divergence in M beta 1 is due to multiple changes that occur throughout the polypeptide chain. The overall level of expression of M beta 1 is low, and is restricted to those tissues (bone marrow, spleen, developing liver and lung) that are active in hematopoiesis in the mouse. We predict that the M beta 1 isotype is functionally specialized for assembly into the mammalian marginal band.

Sequence and expression of the chicken beta 3 tubulin gene. A vertebrate testis beta-tubulin isotype.

We report the determination of the complete DNA sequence for c beta 3, a chicken beta-tubulin gene which we show to be the dominant beta-tubulin expressed in testis. Like all previously studied vertebrate beta-tubulin genes, the gene is divided into four exon sequences interrupted by three intervening sequences (located between amino acids 19 and 20, within codon 56, and within codon 93). Analysis of the program of expression of this gene indicates that it encodes the dominant chicken testis beta-tubulin, although it is also expressed at lower levels in a wide variety of cell and tissue types. Comparison of the predicted polypeptide sequence for c beta 3 with four other available chicken beta-tubulin genes confirms our earlier suggestion that within an otherwise conserved framework, sequences within two variable region domains serve to define specific beta-tubulin polypeptide isotypes. The data indicate that the c beta 3 gene encodes a unique beta-tubulin isotype which diverges from the dominant neuronal beta-tubulin isotype in 18 of 445 residues (4%). Although the protein coding regions of the c beta 3 gene are highly homologous to the chicken c beta 1, c beta 2, c beta 4, and c beta 5 genes previously reported by us, no significant sequence homology with these previously analyzed genes is discernible in the 5'- or 3'-untranslated region sequences, in the intervening sequences, or in the presumptive transcriptional promoter sequences.

Tubulin isotypes: generation of diversity in cells and microtubular organelles.

Diversity of tubulin isotypes is illustrated by consideration of the beta-tubulin isotypes of higher plants and the eukaryotic microbe, Physarum polycephalum, and by the alpha-tubulin isotypes of the protozoan, Trypanosoma brucei. The carrot plant expresses six, well-defined beta-tubulin isotypes that possess characteristic two-dimensional gel coordinates. These six beta-tubulin isotypes are differentially expressed during development of the flowering plant. In a similar manner, Physarum expresses three separate beta-tubuli isotypes during its life cycle; of the two beta 1 isotypes, one is expressed solely in the myxamoeba whilst the other is expressed both in the myxamoeba and in the plasmodium. A further beta-tubulin isotype, beta 2, is expressed only in the plasmodium. In carrot and in Physarum the generation of beta-tubulin diversity appears, in the main, to be generated by the differential expression of a beta-tubulin multi-gene family. However, tubulin isotypes can also be generated by post-translational modifications and T. brucei utilizes two different modifications within one cell. First, the primary translation product, the alpha 1-tubulin isotype, can be acetylated to produce the alpha 3 isotype. Second, both the alpha 1 and alpha 3 isotypes appear to exist in both tyrosinated and detyrosinated forms. The generation of these alpha-tubulin isotypes within the same cell and their presence in particular cellular domains, modulated throughout the cell cycle, reveals a complex relationship between alpha-tubulin isotypes produced by post-translational modifications and the dynamics of microtubule construction.

Multiple forms of tubulin in the cilia and cytoplasm of Tetrahymena thermophila.

Most higher eukaryotic tubulins are separated into alpha- and beta-tubulin when electrophoresed in NaDodSO4- denaturing gels, while many lower eukaryotic tubulins are poorly resolved under these conditions, which include a stacking gel (pH 6.80) and a separating gel (pH 8.80). By lowering the pH of the separating gel to 8.25, we have found that tubulin isolated from the protozoan Tetrahymena thermophila is resolved by one-dimensional polyacrylamide gel electrophoresis into two alpha-tubulins and one beta-tubulin. Moreover, at least five alpha- and two beta-tubulin isotypes are identified in Tetrahymena by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis. Three of these alpha isotypes and one beta isotype are found specifically in ciliary microtubules, while the other two isotypes are found only in the cytoplasmic tubulin pool that was isolated and induced to self-assemble into microtubules in vitro. Peptide mapping by limited proteolytic digestion indicates that the tubulins are closely related. Possible mechanisms for the generation and selection of these tubulin isotypes are discussed.

Five mouse tubulin isotypes and their regulated expression during development.

We describe five mouse tubulin cloned cDNAs, two (M alpha 1 and M alpha 2) that encode alpha-tubulin and three (M beta 2, M beta 4, and M beta 5) that encode beta-tubulin. The sequence of these clones reveals that each represents a distinct gene product. Within the sequence common to the two alpha-tubulin cDNAs, the encoded amino acids are identical, though the 3' noncoding regions are wholly dissimilar. In contrast, the three beta-tubulin cDNAs show considerable carboxy-terminal heterogeneity. Two of the beta-tubulin isotypes defined by the cloned sequences are absolutely conserved between mouse and human, and all three beta-tubulin isotypes are conserved between mouse and rat. This result implies the existence of selective constraints that have maintained sequence identity after species divergence. This conclusion is reinforced by the near identity between a third mouse beta-tubulin isotype and a chicken beta-tubulin isotype. The significance of the interspecies conservation of tubulin isotypes is discussed in relationship to microtubule function. We have used non-cross-hybridizing 3' noncoding region probes from the five cloned mouse tubulin cDNAs to study the developmental expression of each isotype in various mouse tissues. M alpha 1 and M beta 2 are expressed in an approximately coordinate fashion, and their transcripts are most abundant in brain and lung. M alpha 2 and M beta 5 are ubiquitously expressed and to a similar extent in each tissue, with the greatest abundance in spleen, thymus, and immature brain. In contrast, M beta 4 is expressed exclusively in brain. Whereas the expression of the latter isotype increases dramatically during postnatal development, transcripts from all four other tubulin genes decline from maximum levels at or before birth. Tissue-specific development changes in the abundance of tubulin isotype-specific mRNAs are discussed in relationship to organogenesis in the mouse.

Apparent gene conversion between beta-tubulin genes yields multiple regulatory pathways for a single beta-tubulin polypeptide isotype.

We have determined the complete nucleotide sequences of two chicken beta-tubulin genes, beta 1 and beta 2. These genes display an unusual pattern of segmental homology which indicates that they originally arose by gene duplication and have subsequently coevolved by a process that included localized gene conversion or intergenic recombination. Since the beta-tubulin polypeptides encoded by the two genes are virtually identical (99.5%), particularly in the major beta-tubulin isotype defining regions, they almost certainly constitute a single isotypic class of beta tubulin. However, the regulatory properties of the two genes are highly divergent as indicated by analysis of their patterns of expression in different chicken cell types. beta 1 is the major transcript detected in skeletal muscle myoblasts, whereas beta 2 is the major beta-tubulin transcript in cultured sympathetic neurons. The existence of these two genes appears to derive from a regulatory requirement whereby the expression of a single tubulin isotype is mediated through different regulatory programs in development and differentiation. These results thus provide direct experimental support for the hypothesis that gene conversion and intergenic recombination play an important role in evolution by uncoupling the evolution of structural genes from the regulatory sequences which control them.

Identification of two human beta-tubulin isotypes.

The sequence of a human beta-tubulin cDNA clone (D beta-1) is described; our data revealed 95.6% homology compared with the sequence of a human beta-tubulin processed pseudogene derived by reverse transcription of a processed mRNA (Wilde et al., Nature [London] 297:83-84, 1982). However, the amino acid sequence encoded by this cDNA showed less homology with pig and chicken beta-tubulin sequences than the latter did to each other, with major divergence within the 15 carboxy-terminal amino acids. On the other hand, an independently isolated, functionally expressed genomic human beta-tubulin sequence (5 beta) possessed a very high degree of homology with chicken and pig beta-tubulins in this region. Thus, human cells appear to contain two distinct beta-tubulin isotypes. Both the intact beta-tubulin cDNA clone and a subclone containing only the 3' untranslated region detected two mRNA species in HeLa cells; these mRNAs were 1.8 and 2.6 kilobases long and were present in about equal amounts. Two independently subcloned probes constructed from the 3' untranslated region of the 5 beta genomic sequence also detected a 2.6-kilobase beta-tubulin mRNA. However, the 3'-untranslated-region probes from the cDNA clone and the genomic sequence did not cross-hybridize. Thus, at least two human beta-tubulin genes, each specifying a distinct isotype, are expressed in HeLa cells, and the 2.6-kilobase mRNA band is a composite of at least two comigrating beta-tubulin mRNAs.

Identification of Two Human β-Tubulin Isotypes

The sequence of a human beta-tubulin cDNA clone (D beta-1) is described; our data revealed 95.6% homology compared with the sequence of a human beta-tubulin processed pseudogene derived by reverse transcription of a processed mRNA (Wilde et al., Nature [London] 297:83-84, 1982). However, the amino acid sequence encoded by this cDNA showed less homology with pig and chicken beta-tubulin sequences than the latter did to each other, with major divergence within the 15 carboxy-terminal amino acids. On the other hand, an independently isolated, functionally expressed genomic human beta-tubulin sequence (5 beta) possessed a very high degree of homology with chicken and pig beta-tubulins in this region. Thus, human cells appear to contain two distinct beta-tubulin isotypes. Both the intact beta-tubulin cDNA clone and a subclone containing only the 3' untranslated region detected two mRNA species in HeLa cells; these mRNAs were 1.8 and 2.6 kilobases long and were present in about equal amounts. Two independently subcloned probes constructed from the 3' untranslated region of the 5 beta genomic sequence also detected a 2.6-kilobase beta-tubulin mRNA. However, the 3'-untranslated-region probes from the cDNA clone and the genomic sequence did not cross-hybridize. Thus, at least two human beta-tubulin genes, each specifying a distinct isotype, are expressed in HeLa cells, and the 2.6-kilobase mRNA band is a composite of at least two comigrating beta-tubulin mRNAs.