(3, 4). The analysis, in molecular terms, of the mechanisms underlying this plasticity requires the study of the genes encoding these two enzymes.We have identified (5-7) cDNA clones corresponding to rat and human tyrosine hydroxylases. Here, we describe the isolation of a cDNA clone-pChAT-1-that encodes an active porcine ChoAcTase enzyme. The nucleotide and complete amino acid sequence is reported. ¶ Some structural characteristics of porcine ChoAcTase are discussed, and the sequence is compared with that of Drosophila melanogaster reported by Itoh et al. (8). MATERIALS AND METHODSConstruction of a Randomly Primed cDNA Library in the XgtlO Vector. Total RNA from porcine ventral spinal cord was extracted as described by Lomedico and Saunders (9). Poly(A)+ RNA was purified by oligo(dT)-cellulose chromatography. Random DNA sequences 20-50 nucleotides in length were prepared by sonication and DNase I digestion of calf thymus DNA (10) and used as primers for cDNA synthesis. First-strand cDNA was synthesized from 2.5 ,g of ventral spinal cord poly(A)+ RNA with 30-fold excess of random primer. The second-strand synthesis and following steps were carried out using standard procedures (11, 12). The longest cDNAs [_500 base pairs (bp)] were selected on a 5-20o (wt/vol) sucrose gradient and ligated to the XgtlO vector. The amplified library contained =1.2 x 106 independent recombinant phages.Oligonucleotide Screening. The N-terminal sequence of porcine brain ChoAcTase was determined as described (13). A mixture of oligodeoxynucleotides, each containing eight different chains of 29 nucleotides, was prepared with a Biosearch DNA synthesizer model 8600 by the phosphoramidite method and purified by PAGE. The probes were end-labeled to a minimal specific activity of 8 x 108 cpm/,ug. About 106 recombinant phages were plated at 50,000-70,000 plaques per 13-cm plate, and duplicate filters were prepared. Filters were hybridized at 35°C with oligonucleotides in 6x SSC (lx SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7.0), 5x Denhardt's solution (lx Denhardt's solution = 0.02% Ficoll/0.02% polyvinylpyrrolidone/0.02% bovine serum albumin), 10% (wt/vol) dextran sulfate, 0.05% sodium pyrophosphate, herring sperm DNA at 0.1 mg/ml, and Escherichia coli tRNA at 0.1 mg/ml. Filters were then washed at 35°C, 40°C, and 45°C in 6x SSC containing 0.05% NaDodSO4. Positive clones were isolated after three successive rounds of screening. Phage DNA was prepared as Abbreviations: ChoAcTase, choline acetyltransferase; NVP, 4-(1-naphthylvinyl)pyridine. §To whom reprint requests should be addressed.

A procedure is reported that allows the purification and amino terminal sequencing of pig brain choline acetyltransferase. The enzyme (present in extremely low amounts in this tissue) is eluted together with its antibody from an affinity column by a mild pH shift and the resulting enzyme-antibody complex separated by gel electrophoresis. The band corresponding to the enzyme is electroeluted from the gel using volatile solutions allowing the direct determination of the amino acid composition and partial sequence. The first 11 residues are: Pro-Ile-Leu-Glu-Lys-Thr-Pro-Pro-Lys-Met-Ala.