ACTIN-ENCODING CDNAS AND GENE-EXPRESSION DURING THE INTERMOLT CYCLE OF THE BERMUDA LAND CRAB GECARCINUS-LATERALIS

Two actin-encoding cDNAs (act1 and act2) from Gecarcinus lateralis hav e been sequenced or partially sequenced and the corresponding proteins deduced. The act1 cDNA has a complete ORF; the act2 cDNA lacks most o f the 5' end of the coding region. The nucleotide (nt) sequences of bo th clones are very similar to act sequences of many organisms, the mos t closely related being from another arthropod, the silkmoth Bombyx mo ri. The proteins Act1 and Act2 are more similar to vertebrate cytoplas mic actin isoforms (p-actins) than to vertebrate muscle actins (alpha- actins); they are also more similar to animal actins than to those of fungi or plants. Codon usage is strongly biased toward C or G in the t hird position. The deduced number of amino acid (aa) residues and calc ulated M(r) for Act1 are 376 aa and 41.94 kDa, respectively. The deduc ed aa sequence of Act1 is very similar to those of muscle actins of B. mori and Drosophila melanogaster. Southern blots indicated seven to e leven act genes in the crab genome. Northern blots probed with a segme nt from the 3' UTR of act1 showed a single band of approx. 1.6 kb in p oly(A)(+)mRNAs from epidermis, limb bud or claw muscle and in total RN As from ovary and gill, and two bands of approx. 1.6 and 1.8 kb in tot al RNA from midgut gland. Western blots of one-dimensional gels of pro teins from the four layers of the exoskeleton, epidermis, limb buds an d claw muscle were probed with a monoclonal Ab against chicken gizzard actin; tissue- and stage-specific changes in actin content were obser ved. The presence of several isoforms, and differences in their number and occurrence at various stages of the intermolt cycle, were detecte d on Western blots of two-dimensional gels.