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Molecular cloning and amino acid sequence of brain L-glutamate decarboxylase.

ABSTRACT: We used specific polyclonal antibodies against L-glutamate decarboxylase (GAD) to screen a mouse brain cDNA library that was constructed in the expression vector lambda gt11. We obtained 1.5 x 10(6) 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 lambda GAD-Y1089 showed good GAD enzyme activity as determined by both CO2 and gamma-aminobutyric acid methods. The GAD clone thus obtained contains GAD cDNA of approximately 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.

SUBMITTER: Huang WM

PROVIDER: S-EPMC54982 | biostudies-other | 1990 Nov

REPOSITORIES: biostudies-other

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Molecular cloning and amino acid sequence of brain L-glutamate decarboxylase.

Huang W M WM Reed-Fourquet L L Wu E E Wu J Y JY

Proceedings of the National Academy of Sciences of the United States of America 19901101 21

We used specific polyclonal antibodies against L-glutamate decarboxylase (GAD) to screen a mouse brain cDNA library that was constructed in the expression vector lambda gt11. We obtained 1.5 x 10(6) 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 lambda GAD ...[more]

PMID: 2236059

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Project description:Glutamate decarboxylase (GAD) pathway (GDP) is a major acid resistance mechanism enabling microorganisms' survival in low pH environments. We aimed to study the molecular evolution and population genetics of GDP in Lactic Acid Bacteria (LAB) to understand evolutionary processes shaping adaptation to acidic environments comparing species where the GDP genes are organized in an operon structure (Levilactobacillus brevis) versus lack of an operon structure (Lactiplantibacillus plantarum). Within species molecular population genetic analyses of GDP genes in L. brevis and L. plantarum sampled from diverse fermented food and other environments showed abundant synonymous and non-synonymous nucleotide diversity, mostly driven by low frequency changes, distributed throughout the coding regions for all genes in both species. GAD genes showed higher level of replacement polymorphism compared to transporter genes (gadC and YjeM) for both species, and GAD genes that are outside of an operon structure showed even higher level of replacement polymorphism. Population genetic tests suggest negative selection against replacement changes in all genes. Molecular structure and amino acid characteristics analyses showed that in none of the GDP genes replacement changes alter 3D structure or charge distribution supporting negative selection against non-conservative amino acid changes. Phylogenetic and between species divergence analyses suggested adaptive protein evolution on GDP genes comparing phylogenetically distant species, but conservative evolution comparing closely related species. GDP genes within an operon structure showed slower molecular evolution and higher conservation. All GAD and transporter genes showed high codon usage bias in examined LAB species suggesting high expression and utilization of acid resistance genes. Substantial discordances between species, GAD, and transporter gene tree topologies were observed suggesting molecular evolution of GDP genes do not follow speciation events. Distribution of operon structure on the species tree suggested multiple independent gain or loss of operon structure in LABs. In conclusion, GDP genes in LABs exhibit a dynamic molecular evolutionary history shaped by gene loss, gene transfer, negative and positive selection to maintain its active role in acid resistance mechanism, and enable organisms to thrive in acidic environments.

Dataset Information

Molecular cloning and amino acid sequence of brain L-glutamate decarboxylase.

Publications

Molecular cloning and amino acid sequence of brain L-glutamate decarboxylase.

Similar Datasets

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