Identification of a novel membrane-associated protein expressed in neurons of the squid and rodent nervous systems.

In a previous communication, we reported the isolation of a novel cDNA clone (pA6) from a library constructed from squid axonal mRNAs. The partial cDNA clone contained a unique open reading frame that encoded 84 amino acids and was complementary to a moderately abundant mRNA approximately 550-600 nucleotides in length [Chun et al., J. Neurosci. Res. 49 (1997) 144-153]. In this report, we identify the pA6 gene product, and characterize its expression in the squid and rodent brain. Results of immunoblot analyses conducted in squid, using a polyclonal antibody raised against a synthetic peptide corresponding to the C-terminus of the putative protein, established the presence of two pA6 immunoreactive proteins of approximately 14 kDa and 26 kDa in size. In contrast, mouse brain contained only a single 26-kDa immunoreactive species. In both the squid and mouse brain, the expression of pA6 appears highly selective, being detected in certain neurons but not in non-neuronal cells, as judged by both in situ hybridization and immunocytochemistry. Findings derived from light microscopic, double-label immunohistofluorescence studies indicate that pA6 protein co-localizes with prohibitin, a mitochondrial marker protein. Consistent with these results, electron microscopy localized pA6 immunoreactivity to several membrane compartments to include the outer membrane of mitochondria, as well as to the smooth endoplasmic reticulum and tubulovesicles in dendrites, axons, and axon terminals of neurons in the rat brain. Taken together, these findings indicate that pA6 is a novel, membrane-associated protein, which is expressed in the distal structural/functional domains of neurons in both the invertebrate and vertebrate nervous systems.

Local synthesis of nuclear-encoded mitochondrial proteins in the presynaptic nerve terminal.

One of the central tenets in neuroscience has been that the protein constituents of distal compartments of the neuron (e.g., the axon and nerve terminal) are synthesized in the nerve cell body and are subsequently transported to their ultimate sites of function. In contrast to this postulate, we have established previously that a heterogeneous population of mRNAs and biologically active polyribosomes exist in the giant axon and presynaptic nerve terminals of the photoreceptor neurons in squid. We report that these mRNA populations contain mRNAs for nuclear-encoded mitochondrial proteins to include: cytochrome oxidase subunit 17, propionyl-CoA carboxylase (EC 6.4.1.3), dihydrolipoamide dehydrogenase (EC 1.8.1.4), and coenzyme Q subunit 7. The mRNA for heat shock protein 70, a chaperone protein known to be involved in the import of proteins into mitochondria, has also been identified. Electrophoretic gel analysis of newly synthesized proteins in the synaptosomal fraction isolated from the squid optic lobe revealed that the large presynaptic terminals of the photoreceptor neuron contain a cytoplasmic protein synthetic system. Importantly, a significant amount of the cycloheximide resistant proteins locally synthesized in the terminal becomes associated with mitochondria. PCR analysis of RNA from synaptosomal polysomes establishes that COX17 and CoQ7 mRNAs are being actively translated. Taken together, these findings indicate that proteins required for the maintenance of mitochondrial function are synthesized locally in the presynaptic nerve terminal, and call attention to the intimacy of the relationship between the terminal and its energy generating system. J. Neurosci. Res. 64:447-453, 2001. Published 2001 Wiley-Liss, Inc.

An association between a microsatellite polymorphism at the DRD5 gene and the liability to substance abuse: pilot study.

We have conducted a population-based association study of substance abuse and a microsatellite at the dopamine D5 receptor locus (DRD5) in a sample of European-American males and females with substance dependence (SA) or without any psychiatric disorder. Overrepresentation of the most frequent allele (148 bp) was found in males in the SA group (OR = 2.2, P = .02); this finding was reproduced in females (OR = 5.4, p < .001). The difference in the frequencies of this allele between SA males and SA females was statistically significant. The genotype coded in accordance with the dose of this allele correlated with substance abuse liability in males and females (stronger in females) and with novelty seeking in females. There was no evidence of correlation between the genotypes of spouses that could be induced by assortative mating for the liability to substance abuse. The data suggest that the DRD5 locus is involved in the variation and sex dimorphism of substance abuse liability.

Molecular cloning and characterization of a novel mRNA present in the squid giant axon.

Previously, we reported the presence of a heterogeneous population of mRNAs in the squid giant axon. The construction of a cDNA library to this mRNA population has facilitated the identification of several of the constituent mRNAs which encode several cytoskeletal and motor proteins as well as enolase, a glycolytic enzyme. In this communication, we report the isolation of a novel mRNA species (pA6) from the axonal cDNA library. The pA6 mRNA is relatively small (550 nucleotides in length) and is expressed in both nervous tissue and skeletal muscle. The axonal localization of pA6 mRNA was unequivocally established by in situ hybridization histochemistry. The results of quantitative RT-PCR analysis indicate that there are 1.8 x 10(6) molecules of pA6 mRNA (approximately 0.45 pg) in the analyzed segment of the giant axon and suggest that the level of pA6 mRNA in the axonal domain of the giant fiber system might be equal to or greater than the level present in the parental cell soma. Sequence analysis of pA6 suggests that the mRNA encodes an integral membrane protein comprising 84 amino acids. The putative protein contains a single transmembrane domain located in the middle of the molecule and a phosphate-binding loop situated near the N terminus. The C-terminal region of the protein contains two potential phosphorylation sites. These four structural motifs manifest striking similarity to domains present in the ryanodine receptor, raising the possibility that pA6 represents a cephalopod intracellular calcium release channel protein.

Differential compartmentalization of mRNAs in squid giant axon.

Previously, we reported that the squid giant axon contains a heterogeneous population of mRNAs that includes beta-actin, beta-tubulin, kinesin, neurofilament proteins, and enolase. To define the absolute levels and relative distribution of these mRNAs, we have used competitive reverse transcription-PCR to quantify the levels of five mRNAs present in the giant axon and giant fiber lobe (GFL), the location of the parental cell soma. In the GFL, the number of transcripts for these mRNAs varied over a fourfold range, with beta-tubulin being the most abundant mRNA species (1.25 x 10(9) molecules per GFL). Based on transcript number, the rank order of mRNA levels in the GFL was beta-tubulin > beta-actin > kinesin > enolase > microtubule-associated protein (MAP) H1. In contrast, kinesin mRNA was most abundant in the axon (4.1 x 10(7) molecules per axon) with individual mRNA levels varying 15-fold. The rank order of mRNA levels in the axon was kinesin > beta-tubulin > MAP H1 > beta-actin > enolase. The relative abundance of the mRNA species in the axon did not correlate with the size of the transcript, nor was it directly related to their corresponding levels in the GFL. Taken together, these findings confirm that significant amounts of mRNA are present in the giant axon and suggest that specific mRNAs are differentially transported into the axonal domain.

Characterization of squid enolase mRNA: sequence analysis, tissue distribution, and axonal localization.

Enolase is a glycolytic enzyme whose amino acid sequence is highly conserved across a wide range of animal species. In mammals, enolase is known to be a dimeric protein composed of distinct but closely related subunits: alpha (non-neuronal), beta (muscle-specific), and gamma (neuron-specific). However, little information is available on the primary sequence of enolase in invertebrates. Here we report the isolation of two overlapping cDNA clones and the putative primary structure of the enzyme from the squid (Loligo pealii) nervous system. The composite sequence of those cDNA clones is 1575 bp and contains the entire coding region (1302 bp), as well as 66 and 207 bp of 5' and 3' untranslated sequence, respectively. Cross-species comparison of enolase primary structure reveals that squid enolase shares over 70% sequence identity to vertebrate forms of the enzyme. The greatest degree of sequence similarity was manifest to the alpha isoform of the human homologue. Results of Northern analysis revealed a single 1.6 kb mRNA species, the relative abundance of which differs approximately 10-fold between various tissues. Interestingly, evidence derived from in situ hybridization and polymerase chain reaction experiments indicate that the mRNA encoding enolase is present in the squid giant axon.

Kinesin mRNA is present in the squid giant axon.

Recently, we reported the construction of a cDNA library encoding a heterogeneous population of polyadenylated mRNAs present in the squid giant axon. The nucleic acid sequencing of several randomly selected clones led to the identification of cDNAs encoding beta-actin and beta-tubulin, two relatively abundant axonal mRNA species. To continue characterization of this unique mRNA population, the axonal cDNA library was screened with a cDNA probe encoding the carboxy terminus of the squid kinesin heavy chain. The sequencing of several positive clones unambiguously identified axonal kinesin cDNA clones. The axonal localization of kinesin mRNA was subsequently verified by in situ hybridization histochemistry. In addition, the presence of kinesin RNA sequences in the axoplasmic polyribosome fraction was demonstrated using PCR methodology. In contrast to these findings, mRNA encoding the squid sodium channel was not detected in axoplasmic RNA, although these sequences were relatively abundant in the giant fiber lobe. Taken together, these findings demonstrate that kinesin mRNA is a component of a select group of mRNAs present in the squid giant axon, and suggest that kinesin may be synthesized locally in this model invertebrate motor neuron.

beta-Actin and beta-Tubulin are components of a heterogeneous mRNA population present in the squid giant axon.

Previously, we have reported that the squid giant axon contains a heterogeneous population of polyadenylated mRNAs, as well as biologically active polyribosomes. To define the composition of this unique mRNA population, cDNA libraries were constructed to RNA obtained from the axoplasm of the squid giant axon and the parental cell bodies located in the giant fiber lobe. Here, we report that the giant axon contains mRNAs encoding beta-actin and beta-tubulin. The axonal location of these mRNA species was confirmed by in situ hybridization histochemistry, and their presence in the axoplasmic polyribosome fraction was demonstrated by polymerase chain reaction methodology. Taken together, these findings establish the identity of two relatively abundant members of the axonal mRNA population and suggest that key elements of the cytoskeleton are synthesized de novo in the squid giant axon.

Isolation and structural characterization of the bovine tyrosine hydroxylase gene.

A bovine tyrosine hydroxylase (TH) cDNA probe was used to screen a charon 30 genomic library. Screening of approximately 1 million recombinant phage resulted in the identification of one clone, lambda B1, containing the entire bovine TH gene. Results derived from restriction endonuclease mapping and sequence analysis reveal that the bovine gene contains 13 exons spanning approximately 7 kb of genomic DNA. Determination of the transcription initiation site indicates that the TH gene has a 5' untranslated region of 27 bp. A TATA-box sequence is located between positions-29 and -24 from the transcription initiation site and a cyclic AMP regulatory element (CRE) between-45 and -38. Although the TH gene appears to be glucocorticoid responsive in vitro, no regions bearing identity to the consensus sequence for the glucocorticoid regulatory element (GRE) were detected within approximately 1.5 kb of 5' flanking sequence. A cross-species comparison of the 5' flanking sequences of the bovine, rat, and human TH genes reveals strong sequence and positional conservation of seven sequence elements. An analysis of the nucleotide sequence within these elements reveals similarity to the consensus sequences reported for known cis-acting regulatory elements and transcription factor binding sites, suggesting that they may play a role in the regulation of TH gene expression.

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.

The complete nucleotide sequence and structure of the gene encoding bovine phenylethanolamine N-methyltransferase.

A cDNA clone for bovine adrenal phenylethanolamine N-methyltransferase (PNMT) was used to screen a Charon 28 genomic library. One phage was identified, designated lambda P1, which included the entire PNMT gene. Construction of a restriction map, with subsequent Southern blot analysis, allowed the identification of exon-containing fragments. Dideoxy sequence analysis of these fragments, and several more further upstream, indicates that the bovine PNMT gene is 1,594 base pairs in length, consisting of three exons and two introns. The transcription initiation site was identified by two independent methods and is located approximately 12 base pairs upstream from the ATG translation start site. The 3' untranslated region is 88 base pairs in length and contains the expected polyadenylation signal (AATAAA). A putative promoter sequence (TATA box) is located about 25 base pairs upstream from the transcription initiation site. Computer comparison of the nucleotide sequence data with the consensus sequences of known regulatory elements revealed potential binding sites for glucocorticoid receptors and the Sp1 regulatory protein in the 5' flanking region of the gene. Additionally, comparison of the sequence of the exons of the PNMT gene with cDNA sequences for other enzymes involved in biogenic amine synthesis revealed no significant homology, indicating that PNMT is not a member of a multigene family of catecholamine biosynthetic enzymes.

Complexity of nuclear and polysomal RNA from squid optic lobe and gill.

The sequence complexity of nuclear and polysomal RNA from squid optic lobe and gill was measured by RNA-driven hybridization reactions with single-copy [3H]DNA. At saturation, brain nuclear and polysomal RNAs were complementary to 22.8 and 7.9% of the DNA probe, respectively. Assuming asymmetric transcription, the complexity of nuclear and polysomal RNA was equivalent to 2.5 X 10(8) and 8.8 X 10(7) nucleotides, respectively. Approximately 80-85% of the sequence complexity of brain total polysomal RNA was found in the polyadenylated RNA fraction. In contrast to these findings, nuclear and polysomal RNAs from gill hybridized to 9.1 and 2.9%, respectively, of the single-copy DNA, values that were 2.5-fold lower than those obtained in the CNS. Taken together, the results focus attention on the striking diversity of gene expression in the squid CNS and extend to the cephalopod mollusks the observation that nervous tissue expresses significantly more genetic information than other somatic tissues or organs.

Diversity of gene expression in goldfish brain.

The sequence complexity of nuclear and polysomal RNA from goldfish brain and kidney was measured by RNA-driven hybridization reactions with single-copy [3H]DNA. At saturation, brain nuclear and polysomal RNA were complementary to 23.2 and 6.7% of the DNA probe, respectively. In contrast to these findings, nuclear and polysomal RNA from kidney hybridized to 16.1 and 3.1% of the single-copy DNA, values that were significantly lower than that obtained in the CNS. Taken together, the results focus attention on the striking diversity of gene expression in goldfish brain and extend to lower vertebrates the observation that nervous tissue expresses significantly more genetic information than other somatic tissues or organs.

Changes in gene expression during postnatal development of the rat cerebellum.

The base sequence complexity of total and polysomal poly(A +)RNA from rat cerebellum was measured during postnatal development by RNA-DNA hybridization. At saturation, total and polysomal poly(A +)RNA from neonate cerebellum hybridized to 12.7% and 5.0% of the single-copy genomic DNA, respectively. Assuming asymmetric transcription, the sequence complexity of these RNA populations is sufficient to code for greater than 100,000 different gene transcripts. The percentage of single-copy DNA expressed as total and polysomal poly(A +)RNA declined during postnatal development, reaching adult values of 10.0% and 4.1%, respectively. These results indicate that cerebellar maturation is accompanied by significant reductions in the diversity of genetic information expressed in the tissue.

An improved method for the rapid isolation of brain ribonucleic acid.

An improved one-step method for the extraction of RNA from rat brain is described. Fresh or frozen tissue is disrupted in the powerful protein denaturant guanidine thiocyanate, and RNA isolated by ultracentrifugation through CsCl. The procedure is advantageous in that it is relatively simple, is rapid and does not expose the sample to enzyme treatments or repeated organic extractions.