No 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 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...

Taurine is known to be involved in many important physiological functions. Here we report that both in vivo and in vitro the taurine-synthesizing enzyme in the brain, namely cysteine sulfinic acid decarboxylase (CSAD), is activated when phosphorylated and inhibited when dephosphorylated. Furthermore, protein kinase C and protein phosphatase 2C have been identified as the enzymes responsible for phosphorylation and dephosphorylation of CSAD, respectively. In addition, the effect of neuronal depolarization on CSAD activity and 32 P incorporation into CSAD in neuronal cultures is also included. A model to link neuronal excitation and CSAD activation by a Ca 2ϩ -dependent protein kinase is proposed.

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

Cysteine sulfinic acid decarboxylase (CSAD), the rate-limiting enzyme in taurine biosynthesis, appears to be present in the brain in multiple isoforms. Two distinct forms of CSAD, referred to as CSAD I and CSAD II, were obtained on Sephadex G-100 column. CSAD I and CSAD II differ in: (1) the elution profile on Sephadex G-100 column; (2) the sensitivity towards Mn(2+), methione, and other sulfur-containing amino acids, and (3) their immunologic properties. CSAD II has been purified to about 2,500-fold by a combination of column chromatographies and polyacrylamide gel electrophoresis (PAGE). The purity of the enzyme preparation was established as judged from the following observations: (1) a single protein band was observed under various electrophoretic conditions, e.g., 5-20% nondenaturing PAGE, 7% nondenaturing PAGE and 10% SDS-PAGE, and (2) in nondenaturing PAGE, the protein band comigrated with CSAD activity. CSAD II has a molecular weight of 90 kDa and is a homodimer consisting of two 43 +/- 2 kDa subunits. CSAD appears to require Mn(2+) for its maximum activity. Other divalent cations fail to replace Mn(2+) in activation of CSAD activity. However, the precise role of Mn(2+) in the action of CSAD remains to be determined. Copyright 1996 S. Karger AG, Basel