Isolation and nucleotide sequence of a cDNA clone encoding bovine adrenal tyrosine hydroxylase: comparative analysis of tyrosine hydroxylase gene products

Investigations into the structure and mechanisms regulating the expression of the genes involved in catecholamine biosynthesis have led to the isolation of a cDNA coding for bovine adrenal tyrosine hydroxylase (TH). The 1,722 bp cDNA contains the complete coding sequence and 3' untranslated region of the TH mRNA. The nucleotide sequence of the cDNA and the deduced amino acid sequence were compared to those reported for rat and human TH. Bovine TH shares 85% and 84% amino acid sequence identity with that of rat and human TH, respectively. Alignment of the amino acid sequences of rat, bovine, and human TH reveals that 79% of the residues are identical in all three species, indicating a strong evolutionary conservation of enzyme structure. Moreover, three of the four putative phosphorylation sites located in the N-terminal region of TH are conserved in these animal species. There are, however, some interspecies differences in TH gene products. The 3' untranslated region of bovine TH mRNA is 56 and 97 nucleotides shorter than rat and human TH mRNA, respectively. Additionally, the bovine protein is 7 and 6 amino acids smaller than its rat and human homologues. All of the absent amino acid residues of bovine TH are missing from an alanine-rich region in the N-terminal portion of the rat and human proteins (amino acids 51-68). Comparison of the size of bovine and rat TH mRNA and protein by northern blot and immunoblot analyses yielded differences consistent with those predicted from the nucleotide sequence data.     

Cloning of mouse Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKKbeta) and characterization of CaMKKbeta and CaMKKalpha distribution in the adult mouse brain

The Ca(2+)/calmodulin-dependent protein kinase kinases alpha and beta (CaMKKs alpha and beta) are novel members of the CaM kinase family. The CaMKKbeta was cloned from mouse brain. The deduced amino acid sequence shared 96.43% homology with the rat CaMKKbeta. Both the alpha and beta isoforms were widely distributed throughout the adult mouse brain. Additionally, all peripheral tissues examined displayed CaMKK alpha and beta expression.     

Immunohistochemical localization of S100 proteins in dorsal root, sympathetic and enteric ganglia of several mammalian species, including man

The occurrence of S100 proteins in neurons of the mammalian peripheral nervous system is still controversial. This study was designed to investigate this topic in dorsal root ganglia (DRG) and the enteric nervous system (ENS) of several mammalian species (horse, buffalo, cow, sheep, pig, dog, rabbit and rat), as well as in DRG, paravertebral sympathetic ganglia (SG) and ENS of the adult man. Rat embryos of E17 and E19 were also examined. The material was fixed in Bouin's fixative, paraffin-embedded and processed for immunohistochemistry, combined with image analysis, using a panel of mono and polyclonal antibodies against S100alpha, S100beta or S100alpha + beta (referred to here as S100) proteins. In all species examined, strong S100 protein immunoreactivity (IR) was found in satellite glial cells and Schwann cells, which also showed S100alpha and S100beta IR in humans. Furthermore, faint S100 protein IR was observed in a subpopulation of DRG intermediate- and large-sized sensory neurons in humans, buffalo, sheep, and pig. The rat was the only species showing clear S100 and S100beta in neurons, labelling in about 30-35% in adults (small, intermediate and large in size), and about 88% at E17 and 42% at E19, respectively. Weak S100alpha protein IR was observed in most of human SG neurons. In ENS, S100 protein IR was restricted to enteric glial and Schwann cells, with the exception of cow and goat in which a subset of neurons in both the myenteric and submucous plexuses displayed strong S100 protein IR. Neuronal S100alpha IR and glial S100beta IR was found in the human ENS. The present results demonstrate intra- and inter-specific differences in the expression of S100 proteins by neurons of the peripheral nervous system among mammalian species. Furthermore, they also suggest that neuronal S100 protein, at least in humans, consists of both S100alpha and S100beta.     

Molecular cloning and expression of a δ-opioid recetpor from rat brain

We have isolated and characterized a rat δ-opioid receptor. The deduced amino acid sequence (372 aa) closely resembles the murine δ-opioid receptor, DOR-1. In fact, 97% of the amino acid residues are conserved between the two species, while 93% of the nucleic acid residues are identical. A 6 kb mRNA was detected in rat cortex using rat DOR-1 as a probe. When expressed in COS cells, the clone shows high-affinity opioid binding with selectivity for δ-opioids. The rat δ-opioid receptor cDNA clone will be a useful tool for studying the function of δ-opioid receptor in rats. Wiley-Liss, Inc.     

Development of beta-adrenergic receptor and G protein messenger RNA in rat brain

The ontogeny of rat brain beta 1- and beta 2-adrenergic receptor (beta-AR) and G protein messenger RNA (mRNA) was examined by Northern blot analysis using nick-translated cDNA clones for probes. The level of beta 1-AR and beta 2-AR mRNA followed a pattern of development which paralleled that for the receptor binding sites; the levels of mRNA and binding sites were low at day 1 and increased gradually to adult levels by postnatal days 16-25. In contrast, the level of G protein mRNA, including that for Gs alpha, Gi1 alpha, Gi2 alpha, Go alpha and G beta, on postnatal day 1 was equal to or greater than adult levels, increased 40-80% between day 1 and 7 and then decreased to adult values by day 14-25. This developmental time course approximates that reported for the expression of Gs and Gi but not Go and G beta protein levels determined by immunolabeling and toxin catalyzed ADP-ribosylation. The level of beta-actin mRNA was also greater than adult levels on day 1 and then gradually decreased to adult levels by day 14-25. The results indicate that the ontogeny of beta-ARs and G proteins and the relationship between the amount of mRNA and protein are qualitatively different.     

Expression of myosin regulatory light chains in rat brain: characterization of a novel isoform.

We have characterized cDNA clones of mRNAs encoding two distinct isoforms of myosin regulatory light chain expressed in rat brain. One clone, isolated from a cultured astrocyte cDNA library, is derived from a 1200-base mRNA that is expressed at high levels in cultured astrocytes, and at higher levels in the embryonic brain than in the adult brain. The nucleotide sequence of this cDNA is essentially identical to a previously reported cDNA encoding a smooth muscle isoform from rat aorta cells (Taubman et al., J. Cell Biol., 104 (1987) 1505-1515). The second clone hybridized to a 1300-base mRNA that is expressed abundantly in the adult brain and is the predominant species in cultured neuroblasts. Both mRNAs are expressed, to varying extents, in other muscle and nonmuscle tissues. The deduced amino acid sequences of the two isoforms differ in 4 residues out of 171. On the basis of the tissue distribution of their mRNAs and a comparison of identities among the known amino acid sequences of myosin regulatory light chains we suggest that both proteins should be considered as non-muscle isoforms. We conclude that there are at least two isoforms of the myosin regulatory light chain expressed in rat brain and that their expression is under both cell-specific and developmental regulation.     

Subtractive cDNA cloning of RC3, a rodent cortex-enriched mRNA encoding a novel 78 residue protein

A rodent cortex-enriched mRNA, RC3, was identified by screening a rat brain cDNA library with a cortex-minus-cerebellum subtracted cDNA probe. Conceptual translation of RC3 cDNA sequences indicates that the rat and mouse mRNAs encode identical, novel 78 amino acid proteins. The RC3 protein amino terminus contains a cysteine-rich domain similar to those found in snake venom neurotoxins; the carboxyl terminus contains a collagen-like motif that may function in the assembly of RC3 subunits into a multimeric protein. Western blot experiments with an antiserum to a synthetic peptide corresponding to 27 residues of the 78 residue sequence identify an immunoreactive polypeptide with 18 kDa gel mobility that is likely to correspond to the RC3 protein. Northern blot analysis and in situ hybridization experiments show that RC3 mRNA is highly enriched in rat brain, with restricted expression in neuronal subsets primarily in the cortex, striatum, and hippocampus as well as certain nuclei within the thalamus, hypothalamus, the olfactory bulb.     

Structure and expression of the mouse myelin proteolipid protein gene

The gene for the mouse myelin proteolipid protein has been isolated and the seven exons have been sequenced. Since the sequence of a rat proteolipid protein cDNA and partial sequence of the human proteolipid protein gene have been determined, it was possible to demonstrate a very high degree of conservation for the proteolipid protein gene exons among species. While there are some nucleotide changes, the protein coding region of the mouse gene encodes protein that is totally conserved relative to both rat and human proteolipid proteins. The regulatory and noncoding regions of the proteolipid protein gene are also highly conserved. The upstream regulatory and 5'-noncoding region of the gene is 92% homologous to the comparable region of the human proteolipid protein gene, and the 3'-noncoding region of the mouse gene is approximately 90% homologous to a rat proteolipid protein cDNA through 2,200 nucleotides of 3'-noncoding DNA. S1 nuclease protection experiments indicated that the major 5'-end for proteolipid protein mRNAs from mouse, rat, human, or baboon is approximately 147-160 nucleotides upstream from the initial methionine codon of the protein coding region. Other S1 nuclease protection experiments indicated the possible existence of an alternative splice site within exon 3, which may produce mRNA for DM20. This mRNA is approximately 100 nucleotides shorter than that for the proteolipid protein, and it is missing the latter half of exon 3, that is, amino acids 116-150 of the proteolipid protein sequence.     

Molecular cloning and nucleotide sequence of rat brain argininosuccinate lyase cDNA with an extremely long 5'-untranslated sequence: evidence for the identity of the brain and liver enzymes

Argininosuccinate lyase (EC 4.3.2.1) is an enzyme present in the brain of ureotelic animals. Using as a probe rat liver argininosuccinate lyase cDNA, already isolated and sequenced (Amaya, Y., Matsubasa, T., Takiguchi, M., Kobayashi, K., Saheki, T., Kawamoto, S. and Mori, M., J. Biochem., 103 (1988) 177-181), we screened a rat brain cDNA library constructed in the lambda gt11 expression vector and obtained a single cDNA clone. This cDNA clone contained an open reading frame encoding a polypeptide of 461 amino acid residues (predicted Mr = 51,390), a 5'-untranslated sequence of 967 bp and a 3'-untranslated sequence of 74 bp. The length of the 5'-non-coding region of the cDNA seems to be one of the longest among the cDNAs heretofore isolated. A comparison of the brain cDNA sequence (2424 bp) with the corresponding region of the liver cDNA (1574 bp) revealed differences in 5 nucleotides. The brain clone contained A----G and C----G base differences from the hepatic sequence, resulting in amino acid changes from Tyr and Arg in the liver clone, to Cys and Gly in the brain clone, respectively. The other 3 nucleotide differences are silent with respect to the amino acid sequence of the protein. Therefore, the amino acid sequence of the brain argininosuccinate lyase, as deduced from the nucleotide sequence of its cDNA clone, was identical with that of the liver protein, except for two amino acid residues. These minor changes may reflect a microheterogeneity of the argininosuccinate lyase gene. The brain and liver enzymes seem to be encoded by the same structural gene.     

Tissue distribution of rat S-100 alpha and beta subunit mRNAs

Using Southern blot analysis it is demonstrated that S-100 alpha and beta genes were found as single copies in the rat genome. S-100 alpha mRNA was not found at all in rat brain during the developmental period, but it was slightly detectable in muscle and kidney. S-100 beta mRNA was detected in rat brain, and its mRNA level increased during the developmental period. The different localizations of the subunits may be due to the specific expression of each gene in various tissues.     

Molecular and electrophysiological characterization of a allelic variant of the rat alpha 6 GABAA receptor subunit.

A 1.45 kb DNA sequence encoding the rat alpha 6 GABAA receptor subunit (nucleotides 33-1483) was cloned from a Sprague-Dawley rat brain cDNA library by PCR amplification. Dideoxy sequencing of two individual clones revealed that the nucleotide sequence differed at only one basepair (T480-->G) from that published previously. This difference altered the deduced amino acid sequence, producing a conservative amino acid substitution (His121-->Gln). A Gln residue is present at the same location in the bovine alpha 6 subunit. Restriction endonuclease analysis of the total PCR product demonstrated that this variant of the rat alpha 6 subunit was the only allele found in this particular rat brain library, the original allele was not present. These results were further verified by RNAse protection assays performed with RNA isolated from individual rat cerebella. alpha 6, beta 1, and gamma 2S subunits were transiently expressed in L929 cells for electrophysiological analysis. Whole-cell recordings obtained from the cells demonstrated that GABAA receptor channels with the expected GABA and benzodiazepine pharmacology were produced. Excised outside out single channel recordings from the same cells revealed that GABA elicited brief duration openings to a 33 pS main conductance level and to at least one smaller (approximately 21 pS) subconductance level. Thus this allelic variant of rat alpha 6 subunit could assemble with other subunits to form a functional GABAA receptor channel with similar properties to the original allelic form.     

Isolation of a rat adrenal cDNA clone encoding phenylethanolamine N-methyltransferase and cold-induced alterations in adrenal PNMT mRNA and protein

Cold stress is known to increase the synthesis and release of catecholamines in the sympathoadrenal system. Previously, we have demonstrated that cold exposure results in a 3- to 4-fold increase in adrenomedullary tyrosine hydroxylase (TH) activity, which is mediated by concomitant alterations in TH mRNA and protein levels. To further investigate the effects of stress on the expression of the catecholamine biosynthetic enzymes, we have isolated a rat cDNA clone encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT). The cDNA clone is 905 nucleotides in length and contains a single open reading frame corresponding to 270 amino acids. The amino acid sequence predicted from this nearly full-length cDNA is 89% and 86% identical to that of bovine and human PNMT, respectively. Using the rat PNMT cDNA as a hybridization probe, we have measured the effects of cold stress on the relative abundance of adrenomedullary PNMT mRNA. Levels of PNMT protein were also estimated using an immunoblot analysis. As in the case of TH, cold exposure resulted in a rapid and prolonged increase in PNMT mRNA abundance, followed by concomitant increases in PNMT immunoreactivity. However, there appear to be quantitative and qualitative differences in the adaptive response of TH and PNMT to cold stress.     

Developmental regulation of two isoforms of Ca(2+)/calmodulin-dependent protein kinase I beta in rat brain

Subtractive hybridization analysis of region-specific gene expression in brain has demonstrated a mRNA species enriched in rat hypothalamus [K.M. Gautvik, L. de Lecea, V.T. Gautvik, P.E. Danielson, P. Tranque, A. Dopazo, F.E. Bloom, J.G. Sutcliffe, Proc. Natl. Acad. Sci. USA 93 (1996) 8733-8738.]. We here show that this mRNA encodes a Ca(2+)/calmodulin-dependent (CaM) kinase belonging in the CaM kinase I beta subgroup. cDNA analysis showed that this enzyme was differentially spliced into two isoforms (designated beta1 and beta2) with distinct C-termini. The C-terminal of the translated CaM kinase I beta2 protein (38.5 kDa molecular size), contained 25 amino acid residues not present in the beta1 isoform. The two isoforms were differentially developmentally regulated, with the beta1 isoform being present in rat embryos from day 18 and the beta2 isoform being present from day 5 postnatally. In situ hybridization analysis of adult rat CNS showed CaM kinase I beta2 mRNA being enriched in the hypothalamus and the hippocampal formation. Expression was also observed in a number of ventral limbic structures and in the thalamus. Northern blot analysis showed additional expression of multiple beta2 isoforms in heart and skeletal muscle. The human mRNA showed a similar distribution. Our data suggest that the two isoforms of CaM kinase I beta, created by a splicing process occurring within a week around birth, may have distinct pre- and postnatal functions in a distinct set of CNS neurons and excitable tissues.     

Characterization of a cDNA coding for rat glutamic acid decarboxylase

cDNA clones have been isolated for rat glutamic acid decarboxylase (glutamate decarboxylase; EC 4.1.1.15) (GAD) and 3216 bp of the sequence have been determined. This sequence extends the previously reported feline GAD cDNA sequence both in the 5' (67 bp) and 3' (887 bp) directions and contains the polyadenylation signal and tail. The cDNA codes for a 67 kDa mol. wt. protein beginning from the putative initiator methionine found in the feline sequence. Extensive homology to feline GAD was identified at the amino acid level (97% identity) within the coding region. This interspecies homology is high compared to other neurotransmitter synthesizing enzymes and suggests selective pressure to maintain the primary sequence throughout the full length of the protein. Homology is found 5' to the putative initiator methionine. Extensive stretches of homology are also found in the 3' non-coding region. These conserved non-coding regions may play a role in GAD mRNA regulation. The rat cDNA sequence will facilitate investigations into the structure and regulation of the GAD gene.