DEVELOPMENTAL BIOLOGY 85, 505-508 (1981) BRIEF NOTES Change in Expression of the Actin Gene Family during Early Sea Urchin Development GLENN T. MERLINO,’ ROBERT D. WATER, GORDON P. MOORE, AND LEWIS J. KLEINSMITH’ Division of Biological Sciences, The University of Michigan, Ann Arbor, Michigan 48109 Received November 1.4,1980; accepted in revised form February 9, 1981 Expression of the actin gene family was studied during early sea urchin development using in vitro translation and nucleic acid hybridization techniques. Poly(A)+ RNA from four-cell and gastrula-stage S. purpuratus embryos was translated in vitro and the translation products were monitored for the presence of actin. In addition, poly(A)+ and total RNA were prepared from various stages of development, electrophoresed on agarose gels, transferred to diazo- benzyloxymethyl paper, and then hybridized with a cloned actin cDNA probe. Results of these experiments indicate that there is a sharp increase in the level of RNA coding for actin during early development, and that there are two forms of actin specific RNA exhibiting different patterns of control. INTRODUCTION Actin, a highly abundant protein in most eucaryotic cell types, has important functions with respect to cell shape and motility (reviewed by Pollard and Weiling, 19’74; Lazarides and Revel, 1979). Genes coding for actin have been cloned and studied in many systems including yeast, slime mold, chicken, Ox@-ichafoUux, Drosophila, and sea urchins (summarized by Durica et al., 1980; Overbeek et al., 1981). All these organisms, with the exception of yeast, possess families of distinct but re- lated actin genes. The size of these gene families varies from about 3 in Ox@-icha to greater than 15 in Die- tgostelium. Expression of the actin gene family has been studied both at the protein (e.g., Devlin and Emerson, 1980) and RNA levels (e.g., Storti et al., 19’78; Margol- skee and Lodish, 1980). The role of actin during development, where cell types continuously reorient themselves during morphogene- sis, is of particular interest. Actin production is almost certainly carefully regulated throughout early embryo- genesis. In the mouse embryo, for example, actin syn- thesis is selectively stimulated during early develop- ment (Abreu and Brinster, 1978). The sea urchin represents a model developmental system whose gen- eral properties have been extensively described. In Strongylocentrotus purpuratus, the actin coding gene family consists of from 5 to 10 genes, some of which reside near each other in the genome (Durica et al., 1980; Overbeek et al., 1981). A minimum of three actin- like protein spots can be identified on two-dimensional gels (Merlin0 et a2.,1980). In this report we present data i G. T. Merlino’s present address is Laboratory of Molecular Biol- ogy, National Cancer Institute, Bethesda, Md. 20205. * To whom reprint requests should be addressed. which indicate that the level of RNA coding for actin, while very low in oocytes and early cleavage stages, increases sharply between 12 and 24 hr after fertiliza- tion. We discuss possible implications of these results with respect to the regulation of actin gene expression during development. MATERIALS AND METHODS (I) Cloning and Labeling of cDNA Coding for Actin cDNA was synthesized using reverse transcriptase with blastula poly(A)+polysomal RNA as template. The cDNA was rendered double stranded and cloned in the plasmid pBR322 by addition of synthetic Barn Hl link- ers as fully described by Merlin0 et al. (1980). One clone, pSA38, was identified as coding for actin by several criteria, including “positive hybridization/translation” (Harpold et al., 1978). pSA38 plasmid DNA was pre- pared as described by El-Gewely and Helling (1980) and 32Plabeled by the nick-translation method of Rigby et al. (1977). Specific activity was -1 X lo8 cpm/pg. (2) Preparation of Total and Polg(A)+RNA from Embryos Oocytes of Strong&centrotus purpuratus were fer- tilized in vitro and embryos were cultured using stan- dard techniques. Total RNA was prepared by solubili- zation of tissue in 8 M guanidine-HCl, and specific precipitation of RNA in 0.5 vol of ethanol, according to the method of Adams et al. (1977). Poly(A)+ RNA was isolated from embryos by phenol/chloroform/isoamyl alcohol extraction and oligo(dT) affinity chromatogra- phy, as previously described (Merlin0 et al., 1978). For preparation of polysomal poly(A)+ RNA, polysomes 505 0012-1606/81/100505-04$02.00/O Copyright Q 1981 by Academic Press, Inc. All rigbtn of reproduction in my form reserved.