Expression of alpha- and beta-tubulin genes during development of sea urchin embryos

Mature unfertilized eggs of the sea urchin Lytechinus pictus contain multiple alpha-tubulin mRNAs, which range in size from 1.75 to 4.8 kb, and two beta-tubulin mRNAs, 1.8 and 2.25 kb. These mRNAs were found at similar levels throughout the early cleavage stages. RNA gel blot hybridizations showed that prominent quantitative and qualitative changes in tubulin mRNAs occurred between the early blastula and hatched blastula stages. The overall amounts of alpha- and beta-tubulin mRNAs increased two- to fivefold between blastula and pluteus. These increases were due mainly to a rise in a 1.75-kb alpha RNA and a new 2.0-kb beta RNA. Other, minor changes also occurred during subsequent development. All size classes of alpha- and beta-tubulin RNAs in early and late embryos contained poly(A)+ translatable sequences. As reported earlier, some of each of the alpha RNAs, but neither of the beta RNAs, are translated in the egg and a small portion of each of the stored alpha and beta RNAs is recruited onto polysomes within 30 min of fertilization. In the work described here, subsequent development up to the morula stage was accompanied by a gradual recruitment of tubulin mRNAs into polysomes. By the early blastula stage, most of the maternal tubulin sequences were associated with polysomes. In contrast to the gradual recruitment of maternal sequences throughout cleavage, the tubulin mRNAs which appeared at the blastula stage showed no delay in entering polysomes. The exact fraction of each mRNA that was translationally active at later stages varied somewhat among the individual mRNAs. From the differential hybridization patterns of egg, embryo, and testis RNAs to various tubulin cDNA and genomic DNA probes, it is concluded that at least one gene producing maternal alpha mRNA is different from a second one which is expressed only in testis. Each of the three embryonic beta RNAs is encoded by a different beta gene; at least two of these different beta genes are also expressed in testis.     

Sequence of a highly divergent beta tubulin gene reveals regional heterogeneity in the beta tubulin polypeptide

The nucleotide sequence of a chicken genomic DNA segment containing the chicken beta 4 tubulin gene has been determined. The predicted amino acid sequence of beta 4 is surprisingly divergent from that of the chicken beta 2 gene that encodes the dominant neural beta tubulin. beta 4 differs from beta 2 at 36 residue positions and encodes a polypeptide that is four amino acids longer, yielding a divergence of 8.9% between the two beta tubulin isotypes. While many of the amino acid substitutions are conservative, several involve significant alteration in the physiochemical properties of the residue. Furthermore, the amino acid substitution positions are not randomly located within the primary sequence but are distinctly clustered: major divergence occurs in the carboxy-terminal region beyond residue 430 and within the second protein coding exon segments of the genes. In addition, large regions of absolute sequence conservation are also present. Certain sequences within the heterogeneous regions are conserved in other species, indicating that these regions are under positive evolutionary selection pressure and are therefore probably essential for some aspect of beta- tubulin function. These findings strongly suggest that regional amino acid sequence heterogeneity may play an important role in the establishment of functionally differentiated beta tubulin polypeptides.     

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.     

Sequence of chicken c beta 7 tubulin. Analysis of a complete set of vertebrate beta-tubulin isotypes

In chicken, beta-tubulin is encoded by a family of seven genes. We have now isolated and sequenced overlapping cDNA clones corresponding to gene c beta 7 (previously designated c beta 4'), the only chicken beta-tubulin not previously characterized. The inferred amino acid sequence of c beta 7 tubulin is identical with the class I beta-tubulin isotype found in human, mouse and rat. Moreover, c beta 7 is highly expressed in almost all tissue and cell types in chicken, a pattern similar to those of the genes for class I beta-tubulin isotypes in other vertebrates. Comparison of the complete family of chicken beta-tubulin gene sequences reveals that the heterogeneity of beta-tubulin polypeptides encoded in a higher eukaryote is confined to six distinct beta-tubulin isotypes. Five of these are members of evolutionarily conserved isotypic classes (I to V), whereas the sixth represents a divergent erythroid-specific tubulin whose sequence has not been conserved.     

Sequence and expression of the chicken beta 5- and beta 4-tubulin genes define a pair of divergent beta-tubulins with complementary patterns of expression.

We have determined the nucleotide sequence of the chicken beta 5 (c beta 5)-tubulin gene. The gene displayed the coding structure common to all previously studied vertebrate beta-tubulin genes and was divided into four exon sequences interrupted by three intervening sequences (located between codons 19 and 20, within codon 56, and within codon 93). Comparison of the predicted polypeptide sequence encoded by c beta 5 with those of four other available chicken beta-tubulin sequences revealed that c beta 5 encoded a highly divergent beta-tubulin polypeptide isotype which was distinguished from previously known sequences primarily by two discrete variable sequence domains. However, c beta 5 uniquely shared identity in 16 residue positions with another divergent chicken beta-tubulin gene, c beta 4. These common sequences distinguished c beta 4 and c beta 5 from the remaining three chicken beta-tubulin genes. Analysis of the expression of c beta 5 and c beta 4 revealed a strikingly complementary pattern of gene expression: c beta 5 was expressed in a wide variety of cell and tissue types but not in neurons, whereas c beta 4 expression was detected uniquely in neuronal cells. Overall, these findings suggest the existence of two divergent families of beta-tubulin sequences in the chicken and further raise the possibility that the complementary expression of the c beta 4 and c beta 5 genes may fulfill a requirement for the presence of a divergent beta-tubulin polypeptide isotype in all cell types.     

Tubulin isoforms in the brine shrimp, Artemia: primary gene products and their posttranslational modification

The brine shrimp, Artemia, contains 3 alpha- and 2 beta-tubulins as shown by Coomassie Blue staining of two-dimensional gels. In order to study the biosynthetic origins of the isotubulins, we hybridized cloned Drosophila tubulin genes, under stringent conditions, to blots of Artemia DNA and RNA. Southern blot analyses indicate a tubulin gene family of limited complexity. One size class of alpha- and beta-tubulin mRNA at 1800 bases was observed on Northern blots. Fluorograms of Artemia tubulin synthesized in vitro, revealed one alpha- and one beta-tubulin on two-dimensional gels, indicating that each mRNA is translated into one polypeptide and that additional tubulin spots observed on Coomassie-stained two-dimensional gels may arise posttranslationally. Artemia tubulin, which was either purified to homogeneity, or in crude cell-free extracts, was analyzed with a panel of tubulin-specific antibodies. The presence of acetylated tubulin, restricted to one of the three major alpha-tubulin spots on two-dimensional gels, demonstrated that Artemia tubulin diversity is partially generated by posttranslational mechanisms. Artemia tubulin reacted very well with an antibody to tyrosinated tubulin, but there was no, or very little, detectable detyrosinated tubulin unless the purified Artemia tubulin was exposed to carboxypeptidase. The results suggest that all microtubule-dependent events in Artemia, a complex metazoan animal, are accomplished with microtubules composed from a limited repertoire of tubulins and that none of these events require appreciable amounts of detyrosinated tubulin.     

Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation.

Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a beta-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of beta-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal beta-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the beta-tubulin nascent peptide selectively disrupts the regulation of beta-tubulin, but not alpha-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of beta-tubulin mRNA mediated through binding of one or more cellular factors to the beta-tubulin nascent peptide.