Developmentally regulated expression of an exon containing a stop codon in the gene for glutamic acid decarboxylase.

Abstract

In the adult rat brain, the gene for glutamic acid decarboxylase (GAD; L-glutamate 1-carboxy-lyase, EC 4.1.1.15) is expressed predominantly as a 3.7-kilobase transcript. Earlier data showed that embryonic brain expresses an RNA transcript distinct from the adult form; however, the exact structure of this form was not elucidated. Here, transcripts expressed in the embryonic but not the adult brain were cloned and analyzed. These transcripts include an exon not expressed in the adult inserted into the coding sequence. The embryonic exon contains a stop codon that is in-frame with the coding sequence. The exon is found in genomic DNA within the GAD gene where it is flanked by introns with conventional splice sites. On the basis of these structural data, we propose the hypothesis that, early in brain development, transcripts encoding a truncated form of GAD are expressed. The deduced protein cannot function as a decarboxylase because the stop codon in the embryonic exon occurs upstream of the binding site for pyridoxal phosphate, an essential cofactor. Thus, alternative splicing plays a crucial role in the pathway leading to the development of functional GABAergic neurons. The central nervous system-derived cell lines B65 and C6 express a mixture of the adult and embryonic forms of GAD mRNA. They therefore are useful clonal models of central nervous system cells in the early phases of differentiation.

Transient expression of glutamate decarboxylase and γ‐amino butyric acid in embryonic lens fibers of the rat

“…3). Since the GAD67 primary transcript of the embryonic rat may be alternatively spliced (Bond et al, 1990;Szabo et al, 1994), the resulting PCR products could comprise a mixture of cDNAs of 546,626, and 632 bp in length (see Fig. 3).…”

“…At least two GAD enzymes (GAD65 and GAD67) have been identified in the mammalian brain; each is the product of a distinct gene and differs in subcellular localization and developmental expression Bu et al, 1992). The alternatively spliced GAD67 mRNA contains a developmentally regulated exon that is expressed in the embryo (Bond et al, 1990). Interestingly, this exon donates either 80 (ES80) or 86 (ES86) nucleotides to the embryonic mRNA by alternative splicing (Szabo et al, 1994).…”

GAD isoforms exhibit distinct spatiotemporal expression patterns in the developing mouse lens: Correlation with Dlx2 and Dlx5

“…The GAD isoforms are highly homologous at the protein level, but show different affinity to the cofactor pyridoxalphosphate (PLP) and have distinct subcellular localizations. During mouse and rat embryonic development, two additional, alternatively spliced forms are synthesized from the GAD67 gene (Bond et al, 1990;Szabo et al, 1994) encoding the truncated 25-kDa leader (GAD25) and enzymatically active GAD44. GAD25 and GAD44 are transiently expressed during neuronal differentiation (Szabo et al, 1994;Varju et al, 2001Varju et al, , 2002 being more abun-dant in proliferating progenitors and down-regulated in postmitotic neurons concomitant with up-regulation of GAD67 expression (Ma et al, 1993;Behar et al, 1994;Katarova et al, 2000).…”

Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10.

“…In early work, Escherichia coli GAD was crystallized (2) allowing identification of its pyridoxal 5'-phosphate (PLP, or vitamin B6) cofactor binding site, -XaaHis-Lys(E-PLP)-Xaa- (3,4). Multiple forms of GAD (5,6) with distinct tissue expression (1,7), subcellular localization (8)(9)(10), and developmental expression (11,12) have since been reported. Biological functions of GAD and GABA extend beyond regulation of neurotransmission to include effects on the immune system as well as modulation of cell proliferation, protein synthesis, and metabolism (for reviews see refs.…”

“…Recently, a partial human GAD sequence isolated from testis (7) showed the same high homology to the GAD-1 sequences and demonstrated both 2.5-kb and 3.7-kb transcripts in testis. A third isoform, resulting from alternative splicing, is present in embryonic rat brain (12). Finally, another GAD form with little homology to the GAD-1 sequences and coding for a protein of Mr 80,000 has recently been cloned from mouse brain (27).…”