MOLECULAR CHARACTERIZATION OF 4 MEMBERS OF THE ALPHA-TUBULIN GENE FAMILY OF THE BERMUDA LAND CRAB GECARCINUS-LATERALIS

Four alpha-tubulin isoforms recovered from a cDNA library from regener ating limb buds of the Bermuda land crab Gecarcinus lateralis have bee n 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 t heir coding sequences but rather low homology in their non-coding regi ons. 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 alph a-tubulin proteins occurred near the carboxy terminus, as in alpha-tub ulins 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 a mino 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. Nort hern 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 o f both tubulin isoforms appeared to be independently regulated at diff erent stages of the intermolt cycle in both epidermis and limb buds. W estern 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 numbe r of alpha-tubulin isoforms that were expressed. (C) 1997 Wiley-Liss, Inc.dagger.