Primary structure and expression of the gamma 2 subunit of the glutamate receptor channel selective for kainate.

The presence and primary structure of a novel subunit of the mouse glutamate receptor channel, designated as gamma 2, have been revealed by cloning and sequencing the cDNA. The gamma 2 subunit has structural characteristics common to the neurotransmitter-gated ion channel family and shares a high amino acid sequence identity with the rat KA-1 subunit, thus constituting the gamma subfamily of the glutamate receptor channel. Expression of the gamma 2 subunit together with the beta 2 subunit in Xenopus oocytes yields functional glutamate receptor channels selective for kainate.

Role of the carboxy-terminal region of the GluR epsilon2 subunit in synaptic localization of the NMDA receptor channel.

The synaptic localization of the N-methyl-D-aspartate (NMDA) type glutamate receptor (GluR) channel is a prerequisite for synaptic plasticity in the brain. We generated mutant mice carrying the carboxy-terminal truncated GluR epsilon2 subunit of the NMDA receptor channel. The mutant mice died neonatally and failed to form barrelette structures in the brainstem. The mutation greatly decreased the NMDA receptor-mediated component of hippocampal excitatory postsynaptic potentials and punctate immunofluorescent labelings of GluR epsilon2 protein in the neuropil regions, while GluR epsilon2 protein expression was comparable. Immunostaining of cultured cerebral neurons showed the reduced punctate staining of the truncated GluR epsilon2 protein at synapses. These results suggest that the carboxy-terminal region of the GluRepsilon2 subunit is important for efficient clustering and synaptic localization of the NMDA receptor channel.

Impairment of suckling response, trigeminal neuronal pattern formation, and hippocampal LTD in NMDA receptor epsilon 2 subunit mutant mice.

Multiple epsilon subunits are major determinants of the NMDA receptor channel diversity. Based on their functional properties in vitro and distributions, we have proposed that the epsilon 1 and epsilon 2 subunits play a role in synaptic plasticity. To investigate the physiological significance of the NMDA receptor channel diversity, we generated mutant mice defective in the epsilon 2 subunit. These mice showed no suckling response and died shortly after birth but could survive by hand feeding. The mutation hindered the formation of the whisker-related neuronal barrelette structure and the clustering of primary sensory afferent terminals in the brainstem trigeminal nucleus. In the hippocampus of the mutant mice, synaptic NMDA responses and longterm depression were abolished. These results suggest that the epsilon 2 subunit plays an essential role in both neuronal pattern formation and synaptic plasticity.

The structure and expression of neuron-specific enolase gene.

Neuron-specific (gamma gamma) enolase (NSE) is an isoenzyme form of glycolytic enzyme, enolase. We isolated genomic clones for NSE and clarified NSE gene structures. The NSE-gene spanned about 9 kb and consisted of twelve exons and eleven introns. Multiple transcriptional start points were identified by a combination of S1 nuclease mapping and primer extension analysis. In the 5'-flanking region we found a TATA-like sequence TCTATAGGC which was only partially homologous to the consensus sequence, but we did not find a CAAT box. The sequence in the immediate 5'-flanking region was of a relatively high G + C content and contained GC-box-like clusters that did not correspond to the typical GC box. In addition, we found seven classes of the repeated sequences. In the introns 1, 5 and 10 there were tandem repeats (GT)33, (GT)21 and (GT)24, respectively. The 3' end contains a single polyadenylation site and an identifier sequence 2 kb downstream from the poly(A)-addition site. The in vitro cell-free transcription of the truncated genomic DNA fragment using HeLa cell extract showed that the transcription start points have been correctly identified and the putative promoter sequences appear to be functional.

Structure and expression of rat muscle-specific enolase gene.

The structure of rat muscle-specific enolase (beta beta enolase) gene was determined. It comprises 12 exons of various lengths (59-223 bp) spanning about 6 kbp and its exon-intron organization is similar to that of neuron-specific enolase (gamma gamma enolase) gene. A transcriptional start site was identified by a combination of S1 nuclease mapping and primer extension analyses. In the 5'-flanking region we found a TATA-box-like sequence and several MyoD-binding motifs. The in vitro cell free transcription of the truncated genomic DNA fragment using HeLa cell extract showed that the transcription start site has been correctly identified and the promoter sequences work well.

cDNA cloning and nucleotide sequence of rat muscle-specific enolase (beta beta enolase).

The nucleotide sequence of rat muscle-specific enolase cDNA was determined by sequencing three cDNA clones encoding this enolase isozyme. The nearly full-length cDNA consists of 13-bp 5'- and 84-bp 3'-noncoding regions and a poly(A) tail in addition to a 1302-bp coding region encoding a polypeptide composed of 434 amino acid residues. The deduced primary structure of this enolase isozyme is about 80% similar to those determined previously for rat neuron-specific and non-neuronal enolase isozymes. Southern blot analysis suggested strongly the existence of a single copy of the muscle-specific enolase gene per haploid genome. The mRNA for this enolase isozyme was detected in rat skeletal muscle on day 1 after birth and its level increased rapidly during 10-30 days after birth without any change in its size (1500 bases).

Functional expression from cloned cDNAs of glutamate receptor species responsive to kainate and quisqualate.

The complete amino acid sequences of two mouse glutamate receptor subunits (GluR1 and GluR2) have been deduced by cloning and sequencing the cDNAs. Xenopus oocytes injected with mRNA derived from the GluR1 cDNA exhibit current responses both to kainate and to quisqualate as well as to glutamate, whereas oocytes injected with mRNA derived from the GluR2 cDNA show little response. Injection of oocytes with both the mRNAs produces current responses larger than those induced by the GluR1-specific mRNA and the dose-response relations indicate a positively cooperative interaction between the two subunits. These results suggest that kainate and quisqualate can activate a common glutamate receptor subtype and that glutamate-gated ionic channels are hetero-oligomers of different subunits.

Upstream and intron regulatory regions for expression of the rat neuron-specific enolase gene.

Neuron-specific enolase (NSE) occurs in mature neurons and paraneurons. We have isolated the genomic clone coding for rat NSE and clarified its gene structure. In order to analyze the regulatory sequence in the 5'-upstream region and introns, we carried out transient expression experiments of NSE genomic DNA fragments fused to chloramphenicol acetyltransferase (CAT) gene which were transfected into several cultured cells. The used cells were primary cultured rat neurons, PC12, neuroblastoma 35, neuroblastoma 103, C6, primary cultured rat glial cells and HeLa cells. The promoter sequence (190 bp) upstream to the transcription initiation site was important in the expression of CAT gene in these cells. From the experiments with external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in the primary cultured neuron and PC12 cells was found to be localized at upstream 500 bp sequence of the intron 1 and 1.5 kbp upstream sequence of the transcription initiation site. In the upstream important sequences, there were the nearest sequences for AP-1 binding motif, AP-2 binding element, SP-1 binding sequence, cAMP response element, half site of glucocorticoid receptor (GRE) binding sequence, half site of thyroid hormor receptor (TR) or retinoic acid receptor (RAR) binding sequence and MTF-1 binding sequence. Furthermore, Octamer-6 binding motifs also were found. In the intron 1, 5' end upstream 50 bp and downstream 100 bp were the most important sequences. We found the nearest sequences for cAMP response element, E2F binding sequence, early growth response (EGR)-1 binding motif, half site of TCF-1 binding sequence and a neuron-specific element-like sequence in the intron 1.

Cloning and functional expression of a cDNA encoding the mouse beta 2 subunit of the kainate-selective glutamate receptor channel.

The primary structure of the mouse glutamate receptor beta 2 subunit has been deduced by cloning and sequencing cDNA. The beta 2 subunit has structural characteristics common to the subunits of glutamate-gated ion channels. Expression of the cloned cDNA in Xenopus oocytes yields functional glutamate receptor channels selective for kainate.

Changes in levels of translatable mRNA for neuron-specific enolase and non-neuronal enolase during development of rat brain and liver.

Neuron-specific enolase (NSE), and non-neuronal enolase (NNE) which exists in many tissues including liver but is localized in glial cells within the nervous system, were synthesized in the rabbit reticulocyte cell-free translation system programmed with brain mRNAs. The in vitro synthesized NSE and NNE were indistinguishable from the two enzymes purified from rat brains. NSE mRNA activity was found only in brain RNAs, while NNE mRNA activity existed in brain RNAs as well as liver RNAs. In developing brains, the level of translatable NSE mRNA was low at the embryonic stage and at birth, increased rapidly from about 10 days postnatal, and reached the adult level, while that of NNE mRNA was high at the embryonic stage and at birth, followed by a slight decrease then a gradual rise to adult levels. These changes correlated with the developmentally regulated appearance and accumulation pattern of each of the two enzymes. These results suggest that the levels of NSE and NNE are controlled primarily by the level of each of the two translatable mRNAs. In developing livers, only the NNE mRNA activity was detected and its level generally paralleled the changes in the level of NNE.

Complexity of RNA from the neuronal and glial nuclei.

The unique DNA expression value in the brain nuclear RNA was much higher than those from other tissues. The origin of this high complexity of brain nuclear RNA was examined using the [3H]DNA-RNA saturation hybridization technique. RNA was extracted from the isolated neuronal and glial nuclei. The experimental results may indicate that the high complexity in the brain nuclei does not result from the summation of many cell types but only from the neuronal nuclei.

Acetylcholine system in the isolated ventral and dorsal horn neurons from bovine spinal cord.

We have isolated the ventral and dorsal horn neurons from the bovine spinal cord using our isolation procedure and determined choline acetyltransferase, acetylcholinesterase and choline uptake activity in these neurons. Choline acetyltransferase and choline uptake activity were higher in the ventral than in the dorsal horn neurons, showing their cell specificity to some extent, but acetylcholinesterase activity did not show any differences between the two cell types.

Decreased uptake of GABA by dorsal ganglia in methylmercury-treated rats.

The uptake of 14C-GABA and 14C-choline by cerebral cortex slices did not show any change in rats which showed neurological signs 7 days after the last 7 methylmercury injections (10 mg/kg/day). However, 14C-GABA uptake by dorsal ganglia greatly decreased, although 14C-choline uptake did not decrease in dorsal ganglia. 14C-GABA uptake by cerebellum also was somewhat decreased. The degree of inhibition of 14C-GABA uptake by dorsal ganglia increased each day after the commencement of methylmercury injection. The decrease of 3H-GABA uptake by dorsal ganglia of methylmercury-injected rats was confirmed by autoradiography. Autoradiographic and inhibitor studies showed labelled GABA accumulated in the satellite glial cells. In vitro addition of methylmercury (10(-4)-10(-5) M) equally inhibited the uptake of GABA and choline by brain and dorsal ganglia slices. These studies may reveal a possible mechanism of methylmercury neuropathy.

Molecular cloning and the nucleotide sequence of cDNA to mRNA for non-neuronal enolase (alpha alpha enolase) of rat brain and liver.

The nucleotide sequence for alpha alpha enolase (non-neuronal enolase: NNE) of rat brain and liver was determined from recombinant cDNA clones. The sequence was composed of 1722 bp which included the 1299 bp of the complete coding region, the 108 bp of the 5'-noncoding region and the 312 bp of the 3'-noncoding region containing a polyadenylation signal. In addition, the poly(A) tail was also found. A potential ribosome-binding site was located 30 nucleotides upstream to the initiation codon in the 5'-noncoding region. The amino acid sequence deduced from the nucleotide sequence was 433 amino acids in length and showed very high homology (82%) to the amino acid sequence of gamma gamma enolase (neuron-specific enolase: NSE), although the nucleotide sequence showed slightly lower homology (75%). The size of NNE mRNA was approximately 1800 bases by Northern transfer analysis and much shorter than that of NSE mRNA (2400 bases) indicating a short 3'-noncoding region. A dot-blot hybridization and Northern transfer analysis of cytoplasmic RNA from the developing rat brains using a labeled 3'-noncoding region of cDNA (no homology between NSE and NNE) showed a decrease of NNE mRNA at around 10 postnatal days and then a gradual increase to adult age without changes of mRNA size. Liver mRNA did not show any significant change during development.

Partial purification and characterization of messenger RNA coding 14-3-2 protein from rat brain.

14-3-2 Protein (neuron-specific enolase) is a neuron-specific protein. Using a reticulocyte lysate cell-free system for translation of 14-3-2 protein mRNA, we have partially purified this mRNA by several procedures, including formamide sucrose density centrifugation, formamide polyacrylamide gel electrophoresis (PAGE) and polyuridylic acid (poly(U))-Sepharose affinity chromatography. Using mRNA obtained by these procedures, we could increase the translation ratio of 14-3-2 protein synthesized/total soluble protein synthesized to 7.31%. The overall purification was 37.8-fold. The size of 14-3-2 protein mRNA appears to be about 19-20S, because translation activity of mRNA obtained by sucrose density gradient centrifugation or formamide PAGE was the most active in this RNA size.

Selective expression of the glutamate receptor channel delta 2 subunit in cerebellar Purkinje cells.

The primary structure of a putative subunit of the mouse glutamate receptor channel, designated as the delta 2 subunit, has been deduced by cloning and sequencing the cDNA. The delta 2 subunit has four putative transmembrane segments characteristic for neurotransmitter-gated ion channels, and shares 56% amino acid sequence identity with the delta 1 subunit of the mouse glutamate receptor channel and 14-24% identity with the subunits of the AMPA-, kainate- or NMDA-selective glutamate receptor channel. RNA blot and in situ hybridization analyses show that the delta 2 subunit mRNA is localized in cerebellar Purkinje cells. Furthermore, immunoblot and immunohistochemical analyses suggest that the delta 2 subunit protein is actually expressed in vivo in Purkinje neurons. The selective localization of the delta 2 subunit in Purkinje cells may imply a role of the delta 2 subunit in Purkinje cell-specific function such as the cerebellar LTD.

ID sequences in the genes of three brain-specific proteins.

We characterized the brain-specific gene coding for rat S-100 protein beta-subunit and found three "brain identifier (ID)" elements, which have been proposed to regulate the gene expression in rat brain. The nucleotide sequences of these elements corresponded well with that of the consensus ID element and were clearly different from those of "ID-like" elements in rat beta B1-crystallin gene, etc. ID elements were also observed in the flanking regions of rat neuron-specific enolase and cholecystokinin genes, which were expressed in the neuronal cells. Direct repeats were observed in the regions flanking ID elements.

Molecular cloning and the nucleotide sequence of cDNA for neuron-specific enolase messenger RNA of rat brain.

The cDNAs to mRNA for rat gamma gamma enolase (neuron-specific enolase; NSE; EC 4.2.1.11) were isolated from a cDNA library by using differential colony hybridization and a hybrid-selected translation assay. By overlapping of the nucleotide sequences of several cDNA inserts, it was found that they spanned 2232 base pairs (bp) which included 1299 bp of the complete coding region, 68 bp of the 5' noncoding region, and 848 bp of the 3' noncoding region, including a polyadenylylation signal. In addition, the poly(A) tail was also found. The amino acid sequence deduced from the nucleotide sequence was composed of 433 amino acids. Southern blot analysis with a cDNA insert detected one hybridizing fragment in rat genomic DNA digested with several different restriction enzymes. Dot-blot and transfer hybridization analyses of poly(A)+ RNA from developing rat brains showed an increase of NSE mRNA 10-30 days after birth.

gamma-Aminobutyric acid system in isolated dorsal and ventral horn neurons from bovine spinal cord.

In order to search for the relationship between the structure and the function of the nervous system, the spinal cord provides suitable material. We devised a procedure for isolation of large ventral and small dorsal horn neurons. Then we examined the gamma-aminobutyric acid (GABA) system in both neurons isolated using our procedure. Glutamic decarboxylase (GAD) activity in dorsal horn neurons was much higher than that in ventral horn neurons. Further, GABA uptake activity by the dorsal horn neurons was also somewhat higher than that by the ventral horn neurons, although some properties of GABA uptake were found to be almost the same in both neuronal fractions. However, we could not find any difference of GABA-alpha-ketoglutarate transaminase (GABA-T) activity between the dorsal and ventral horn neurons. These results suggest that GAD and GABA uptake may be indicators for cell specificity to some extent.