Partial Cloning and Nucleotide Sequencing of Glutamate Decarboxylase Gene Isoform ۶۵ from Human Brain

Background: Gamma -aminobutyric acid (GABA), a non-protein amino acid acts as an inhibitory neurotransmitter in the central nervous system of mammalians. The glutamate decarboxylase (GAD) is responsible for the conversion of L-glutamate to GABA. The human brain has two isoforms of this enzyme, GAD۶۵ and GAD۶۷ that differ in molecular weight, amino acid sequence, antigenicity, cellular location and interaction by factor of pyridoxal phosphate. The purpose of this study was cloning of gene encoding the human glutamate decarboxylase. Materials and Methods: Total cellular RNA was extracted from human brain tissue and then converted to cDNA. PCR was performed using exclusive primers for gad gene amplification. After purification of PCR product, it was partially cloned successfully in pJET۱.۲ blunt t-vector and was sent for sequencing. Results: The outcomes indicate that only gad gene was cloned partially. The length of human gad gene isoform ۶۵ is ۱۷۵۹ base pair that encodes ۵۸۵ amino acids. The length of partially cloned gad gene in this study was ۳۸۵ base pair. Conclusion: Because obtaining fresh human brain is difficult and amount of mRNA is low, it may not be easy to clone full length of human gad gene. The approach described in this paper may be useful in cloning of other genes for which the corresponding mRNA is present at low levels.

Keywords:

Glutamate decarboxylase , Gamma -aminobutyric acid , Cloning