Cloning and sequencing of c-DNA encoding N-methyl-D-aspartate receptor subunit-1 in the hypothalamus of male sheep

Ana Zuley Ruiz

doi:10.1590/s1516-35982014000400004

Ana Zuley Ruiz

Open Access

https://doi.org/10.1590/s1516-35982014000400004

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Abstract

The objective of the present study was to report the molecular cloning and determination of the sequences N-methyl-D-aspartate subunit-1 receptor (NMDAR-1) in the hypothalamus of sheep. The re-amplified DNA template for NMDA -1 from the hypothalamus of male sheep was cloned using pBluescript-Sk-plasmids (pBSK, 2958 bp). Purified plasmids containing the NMDA receptor c-DNA were sequenced using the dye-terminator chemistry at the University of Nebraska-Lincoln, Nebraska, USA. Results were entered into the National Center for Biotechnology Web site as accession number AY434689. The sequence of Ovis aries (sheep) NMDAR-1 mRNA from hypothalamus of male sheep has 94-97% homology with Homo sapiens, 94-100% homology with Sus scrofa, and 88-90% homology with Rattus norvegicus NMDAR-1 mRNA sequence, and 94-97% homology with Homo sapiens glutamate receptor and transcript variant NR1-2 and NR1-1 mRNA sequence. These results show high evolutionary conservation of NMDA receptor subunit-1 across species.

Highlights

The excitatory amino acids (EAA), aspartate and glutamate, are considered the major endogenous neuroactive substances involved in excitatory neurotransmission in the brain
The NMDA receptor is formed by the subunit N-methyl-D-aspartate subunit-1 receptor (NMDAR-1) in combination with NMDAR-2A through NMDAR-2D
The mRNA of NMDA receptor was sequenced using the template DNA obtained by PCR-based amplification

Summary

Introduction

The excitatory amino acids (EAA), aspartate and glutamate, are considered the major endogenous neuroactive substances involved in excitatory neurotransmission in the brain. Electrophysiological and radioligand studies have shown that the stimulatory effects of EAA are used in a variety of different postsynaptic receptor subtypes. The two main classes of glutamate receptors are ionotropic and metabotropic. Receptors in the ionotropic glutamate receptor classification are composed of: 1) N-methyl-D-aspartic acid (NMDA) receptors; 2) kainic acid (KA) receptors; 3) 2-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid (AMPA) receptors; and 4) amino-4-phosphobutyric acid (L-AP4) receptors. Molecular biological techniques have revealed that the NMDA-receptor-channel-complex comprises two subunits. The NMDA receptor is formed by the subunit NMDAR-1 in combination with NMDAR-2A through NMDAR-2D. Conventional NMDA receptors are composed of obligate NMDAR-1 subunits and of subunits NMDAR-2A to NMDAR-2D (Monaghan et al, 1989; Monyer et al, 1992; Matsuda et al, 2003; Furukawa et al, 2005)

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