DNA sequence and pattern of expression of the sea urchin (Paracentrotus lividus) α‐tubulin genes

Abstract

To study the molecular aspects of the regulation of transcription of a multigene family, we have isolated and sequenced cDNA and genomic clones coding for the alpha-tubulin of the sea urchin Paracentrotus lividus. Two cDNA clones, P alpha 10 and P alpha 4, contain respectively the coding information for 391 C-terminal and for 338 N-terminal amino acids of the 452 residues that constitute the complete protein. They show silent nucleotide substitutions only, suggesting that P alpha 10 and P alpha 4 represent the cloned copies of two allelic gene transcripts, which encode for two alpha-tubulin isoforms with identical amino acid sequence in the region of the overlap. The comparison of the predicted amino acid sequence of the composite P alpha 4-10 and of the mouse M alpha-6 (Villasante et al., Mol Cell Biol 1986; 6:2409-2419) reveals a conservation of 97% between the two polypeptides. By RNA blotting hybridization six major alpha-tubulin transcripts were identified. Two, of 3.5 kb and 2.0 kb, are expressed in the unfertilized eggs and during early cleavage. The other two maternal mRNAs, of 2.4 kb and 1.8 kb, are expressed in both early and late cleavage embryos, but in the intestine the 1.8 kb RNA, which specifically reacted with the 3' specific probe of the P alpha 10 cDNA, is the only transcript detected. Finally, the 1.5 kb and 1.9 kb mRNAs represent the transcription of stage- and tissue-specific genes, respectively. In fact, the former becomes detectable at blastula stage and accumulates during late development, whereas the latter is found in the testis only. The sequence data of the 3' terminus of the alpha-3 genomic clone suggests that it encodes for a divergent alpha-tubulin, and it most probably corresponds to the testis-specific gene.

Chromatin dynamics of the developmentally regulatedP. lividus neural alpha tubulin gene

“…2C) with a peak of acetylation at 24 hrs of development. This dynamics of the PTMs is in agreement with the expression profile of the PlTalpha2 tubulin gene during development, in fact the alpha2 transcript appears only from blastula stage (about 15 hrs of development), accumulating during late developmental stages (Gianguzza et al, 1989).…”

“…We found that specific histone modifications (H3K9Ac, H3K-4me3), which induce an accessible chromatin conformation, occur dynamically at level of core promoter in the same developmental window previously highlighted as the temporal interval in which the PlTalpha2 neural gene is expressed (Gianguzza et al, 1989;Gianguzza et al, 1995).…”

“…1) at level of core promoter, first intron and second intron. This observation suggested that a different chromatin structure, permissive for transcription, was acquired, during the development, by the PlTalpha2 gene and particularly by its core promoter at gastrula stage, in agreement with its developmentally regulated transcriptional activation at this stage (Gianguzza et al, 1989).…”

“…Since transcription of the PlTalpha2 gene is developmentally regulated (Gianguzza et al, 1989), we supposed that its core promoter could be differentially accessible to RNA polymerase during sea urchin development. In order to investigate this aspect, nuclei from P. lividus embryos at both morula (when the gene is off) and gastrula stage (when the gene is switched-on) were treated with increasing amounts of DNaseI and analyzed by Southern blot hybridization.…”

“…Its expression starts from the hatching blastula stage in the apical plate and progressively increases during embryogenesis (Gianguzza et al, 1989), maintaining its localization restricted to the larval nervous system (apical, oral and lateral ganglia and ciliary band; Gianguzza et al, 1995). Accordingly, the deduced encoded protein is a specific terminal product of neural differentiation: it is indeed an axonal glutamylated tubulin isotype, homologous to the corresponding neural mammalian isotype (Casano et al, 1996;Tuszynski et al, 2006).…”