The nucleotide sequences at the 5' end of one actin cDNA and six actin genomic clones from Dictyostelium have been determined. The amino acid sequences derived from the nucleotide sequences show strong conservation for six of the seven genes relative to the NH2-terminal regibn of Physarum actin. The region 5' to the AUG initiating codon is >90% A+T residues in all of the genes.Actin, a ubiquitous protein in eukaryotic cells, is associated with a large number of cellular functions including contraction and motility (1,2 the untranslated region adjacent to the 51 end of the initiating AUG codon of all of the actin genes analyzed is extremely A+T rich. Except for the low G+C content there is little additional sequence homology in this region among the various genes.MATERIALS AND METHODS Labeling of DNA Fragments. Plasmid DNA was digested with appropriate restriction endonucleases and was then heated at 650C for 5 min. The reaction was diluted 1:2 with 15 mM Tris (pH 8.0) and the 5'-terminal phosphate residue was removed by digestion with calf intestine alkaline phosphatase for 45 min at 37'C. The phosphatase was removed either by three phenol/chloroform extractions (in 0.2% sodium dodecyl sulfate/0.8 M LiCl/50 mM Tris, pH 9.5) followed by two ethanol precipitations or by isolation of DNA fragments by electrophoresis on agarose or acrylamide gels. Gel-purified fragments were free of phosphatase activity.Nondenatured DNA fragments were labeled in vitro by using ['y-32P]ATP according to the method of Maxam and Gilbert (10). [,y-32P]ATP (estimated specific activity > 8000 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) was synthesized according to the method of Johnson and Walseth (11). In some cases, DNA restriction fragments were end-labeled by using DNA polymerase I (Klenow fragment) and [a-32P]dXTPs (12). After labeling, the material was precipitated with ethanol, resuspended in restriction buffer, and digested with the appropriate restriction endonuclease. The majority of the labeled and unlabeled fragments were purified by agarose gel electrophoreses. Restriction endonuclease reactions were stopped with gel loading buffer and loaded directly on a flat-bed agarose gel containing 1 ,ug of ethidium bromide per ml and run as described (13). The mobility of the fragment was visualized during the run with short wavelength UV light. After appropriate separation of the fragment, a small trough (extending across the distance of the slot and approximately 0.5-1 cm wide) was cut in the gel immediately to the anode side of the fragment. The agarose was removed and the trough was filled with running buffer. The DNA fragment was isolated by electrophoreses into the trough at 350 V. The movement of the DNA into the trough was visualized by UV light. DNA in the trough was removed periodically (at approximately 1-min intervals) to avoid electrophoresis of the fragment into the agarose at the opposite edge of the trough. Eluted DNA was then extracted once with phenol/chloroform as described above and precipitated twice with ethanol...