Molecular characterization of four members of the α-tubulin gene family of the Bermuda land crabGecarcinus lateralis

Abstract

Four alpha-tubulin isoforms recovered from a cDNA library from regenerating limb buds of the Bermuda land crab Gecarcinus lateralis have been characterized. Two clones (alpha 1 and alpha 2) 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 alpha 1 and alpha 2 was 83.4%; between their predicted amino acid sequences it was 88.9%. The inferred number of amino acid residues for both alpha 1 and alpha 2 was 451, and their calculated molecular weights were 58.29 and 58.45 kDa, respectively. The greatest divergence in the predicted crab alpha-tubulin proteins occurred near the carboxy terminus, as in alpha-tubulins of other organisms. When compared with other species; nucleotide sequences of all four clones showed highest homology to alpha-tubulin genes of an insect (Drosophila melanogaster), while their predicted amino acid sequences were most highly homologous to an alpha-tubulin of a mammal (Rattus norvegicus). Southern blots revealed a total of five to seven alpha-tubulin genes encoded in the G. lateralis genome. Northern blots showed single bands of approximately 2.2 kb with an alpha 1-tubulin probe and 1.9 kb with an alpha 2-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 1D 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 alpha-tubulin isoforms that were expressed.