We used specific polyclonal antibodies against L-glutamate decarboxylase (GAD) to screen a mouse brain cDNA library that was constructed in the expression vector Agt11. We obtained 1.5 X 106 recombinant DNA clones in the mouse brain cDNA library. One of the clones was positively identified as a GAD clone on the basis of the following results: (i) the clone and its secondary and tertiary clones all reacted strongly with anti-GAD antibodies; (ii) the fusion protein obtained from AGAD-Y1089 showed good GAD enzyme activity as determined by both CO2 and y-aminobutyric acid methods. The GAD clone thus obtained contains GAD cDNA of "=2.6 kilobases that has one internal EcoRI site. After GAD cDNA was cut at the EcoRI site, two DNA fragments of about 1.6 and 1.0 kilobases were obtained at the 5' and 3' ends, respectively. The cDNA insert was found to be composed of 2632 base pairs, the translation initiation site was assigned to the methionine codon ATG, and the termination site was found to be TGA (positions 2216-2218). Furthermore, the coding region in 2169 base pairs was found to consist of 723 amino acids. The protein has a molecular weight of 83,207 and contains 83 strongly basic, 108 strongly acidic, 226 hydrophobic, and 221 polar amino acids with an isoelectric point of 5.355. The relationship of this GAD cDNA to other forms of GAD is discussed. y-Aminobutyric acid (GABA) has been established as a major neurotransmitter in the mammalian central nervous system from physiological, biochemical, pharmacological, and morphological studies (1-3). The rate-limiting step in GABA biosynthesis is the decarboxylation of L-glutamic acid by glutamate decarboxylase (GAD; L-glutamate 1-carboxylyase, EC 4.1.1.15) (1, 2) and hence GAD has been used as a specific marker for GABAergic neurons. Although much progress has been made in the identification of GABAergic neurons and their synaptic connectivities (for review, see ref.3), little is known with certainty regarding the regulation of GAD activity or the expression of the GAD gene. This is partially hampered by the existence of multiple forms of GAD, differing in molecular weight (4), kinetic properties (5-7), and hydrophobic properties (7), and the lack ofdetailed structural information about those forms of GAD. In this communication, we describe the cloning of the mouse brain GAD gene and the elucidation of a complete nucleotide sequence and the deduced amino acid sequence as a first step toward addressing these questions. 11 In addition, a comparison of similarities and differences between the mouse brain GAD gene presented in this communication and the feline GAD gene reported previously (8, 9) is included.
MATERIALS AND METHODSConstruction of cDNA Library. A mouse brain cDNA library was constructed in Agtll as described by Young and Davis (10). Briefly, total RNA was extracted from mouse brain, and poly(A)+ RNA was prepared by oligo(dT)-cellulose chromatography (11). Poly(A)+ RNA was reverse transcribed into double-stranded cDNA, which was inserted into the EcoRI sit...
