Actin-encoding cDNAs and gene expression during the intermolt cycle of the Bermuda land crab Gecarcinus lateralis

Abstract

Two actin-encoding cDNAs ( act1 and act2) from Gecarcinus lateralis have been sequenced or partially sequenced and the corresponding proteins deduced. The actl cDNA has a complete ORF; the act2 cDNA lacks most of the 5′ end of the coding region. The nucleotide (nt) sequences of both clones are very similar to act sequences of many organisms, the most closely related being from another arthropod, the silkmoth Bombyx mori. The proteins Actl and Act2 are more similar to vertebrate cytoplasmic actin isoforms (β-actins) than to vertebrate muscle actins (α-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 third position. The deduced number of amino acid (aa) residues and calculated M r for Actl are 376 aa and 41.94 kDa, respectively. The deduced aa sequence of Actl is very similar to those of muscle actins of B. mori and Drosophila melanogaster. Southern blots indicated seven to eleven act genes in the crab genome. Northern blots probed with a segment from the 3′ UTR of actl showed a single band of approx. 1.6 kb in poly(A) +mRNAs from epidermis, limb bud or claw muscle and in total RNAs from ovary and gill, and two bands of approx. 1.6 and 1.8 kb in total RNA from midgut gland. Western blots of one-dimensional gels of proteins from the four layers of the exoskeleton, epidermis, limb buds and claw muscle were probed with a monoclonal Ab against chicken gizzard actin; tissue- and stage-specific changes in actin content were observed. The presence of several isoforms, and differences in their number and occurrence at various stages of the intermolt cycle, were detected on Western blots of two-dimensional gels.