Reverse Transcription-coupled Polymerase Chain Reaction Research Articles

Runx Expression Is Mitogenic and Mutually Linked to Wnt Activity in Blastula-Stage Sea Urchin Embryos

BackgroundThe Runt homology domain (Runx) defines a metazoan family of sequence-specific transcriptional regulatory proteins that are critical for animal development and causally associated with a variety of mammalian cancers. The sea urchin Runx gene SpRunt-1 is expressed throughout the blastula stage embryo, and is required globally during embryogenesis for cell survival and differentiation.Methodology/Principal FindingsDepletion of SpRunt-1 by morpholino antisense-mediated knockdown causes a blastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU) incorporation and direct cell counts. Reverse transcription coupled polymerase chain reaction (RT-PCR) studies show that the cell proliferation deficit is presaged by a deficit in the expression of several zygotic wnt genes, including wnt8, a key regulator of endomesoderm development. In addition, SpRunt-1-depleted blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferation is also impeded by knockdown of either wnt8 or cyclinD. Chromatin immunoprecipitation (ChIP) indicates that Runx target sites within 5′ sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late blastula stage. Furthermore, experiments using a green fluorescent protein (GFP) reporter transgene show that the blastula-stage operation of a cis-regulatory module previously shown to be required for wnt8 expression (Minokawa et al., Dev. Biol. 288: 545–558, 2005) is dependent on its direct sequence-specific interaction with SpRunt-1. Finally, inhibitor studies and immunoblot analysis show that SpRunt-1 protein levels are negatively regulated by glycogen synthase kinase (GSK)-3.Conclusions/SignificanceThese results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.

Transcriptional Regulation of Brain-Derived Neurotrophic Factor in the Amygdala during Consolidation of Fear Memory

We have demonstrated previously that brain-derived neurotrophic factor (BDNF) signaling in the amygdala is required for the consolidation of fear memory. This study is designed to characterize the signal cascades by which fear conditioning modulates transcriptional and translational expression of BDNF. Real-time reverse transcription-coupled polymerase chain reaction showed a significant increase in BDNF exon I- and III-containing mRNA in the amygdala of fear-conditioned rats, indicating that fear conditioning was capable of up-regulating BDNF mRNA. Bilateral administration of actinomycin D or anisomycin to the amygdala attenuated conditioning-induced increase in BDNF protein. Inhibitors for N-methyl-d-aspartate (NMDA) receptor, L-type voltage-dependent calcium channel (L-VDCC), adenylyl cyclase, cAMP-dependent protein kinase (PKA), and calcium/calmodulin-dependent kinase IV (CaMKIV) significantly reduced the increase. Moreover, DNA affinity precipitation and chromatin immunoprecipitation assays showed that phosphorylated cAMP response element-binding protein (p-CREB) binding activity in the proximal region of BDNF promoter I and III was significantly increased after fear conditioning. Intra-amygdala administration of cAMP response element decoy DNA before training impaired fear learning. Taken together, these results suggest that calcium influx through NMDA receptors and L-VDCCs during fear conditioning activates PKA and CaMKIV resulting in CREB phosphorylation. The phosphorylated CREB binds to BDNF promoter and up-regulates the expression of BDNF in the amygdala, which helps the consolidation of fear memory.

Cloning, expression and chromosomal assignment of human pleckstrin 2

We report the isolation of human pleckstrin 2 cDNA. The cDNA contains a 1059 bp open reading frame encoding a polypeptide of 353 amino acid residues. The deduced amino acid sequence indicates that pleckstrin 2 contains two pleckstrin homology domains and a DEP (dishvelled, egl-10, and pleckstrin) domain and had a 95% identity with the sequence of mouse pleckstrin 2. Northern blot and a reverse transcription-coupled polymerase chain reaction analysis revealed that pleckstrin 2 mRNA is widely expressed in a variety of cell lines. The chromosomal location of the mouse pleckstrin 2 gene was on the D3 band of chromosome 12, as determined by fluorescence in situ hybridization and the human pleckstrin 2 gene was mapped to chromosome 14q24.1 by a bioinformatics analysis.

Septin 3 gene polymorphism in Alzheimer's disease.

Septin 3 is a novel member of the septin subfamily of GTPase domain proteins that was recently identified in human neuronal cells. These proteins are involved in vesicle trafficking, neurite outgrowth, and neurofibrillary tangle formation; however, the expression and functional role of septin 3 in normal neuronal tissues and as an etiological agent in neurological disorders is currently unclear. To further characterize these parameters, the present study analyzed the expression of three isoforms of septin 3 (A, B, and C) in fetal and adult human brains and polymorphism of the septin 3 exon 11 microsatellite in control, pure Alzheimer's disease (AD), Lewy body variant (LBV) of AD, and Parkinson's disease. Septin 3 mRNAs for isoforms A and B, but not C, were detected in the frontal cortex of fetus and adult human samples, as measured by reverse transcription-coupled polymerase chain reaction. Genotype analyses indicated that polymorphic septin 3 alleles were distributed in two peaks of frequency in both control and disease groups. Categorization of the alleles into short (S) and long (L) types revealed a significant difference between AD patients and controls (p = 0.034 by chi-square test). Furthermore, the S-allele homozygosity was significantly underrepresented in AD compared with control (p = 0.015 by chi-square test). These results suggest that polymorphism in exon 11 of septin 3 may have a determinative role in the pathogenesis of AD.

Differential transcriptional regulation of sulfur assimilation gene homologues in the Saccharomyces carlsbergensis yeast species hybrid.

The allopolyploid yeast Saccharomyces carlsbergensis appears to be a relatively newly formed species hybrid, and therefore constitutes a good model for studying early steps in hybrid speciation. Using reverse transcription-coupled polymerase chain reaction to monitor derepression of the S. carlsbergensis homologues of the sulfur assimilation genes MET14 and MET2, we found that both homologues of these genes are regulated in the same pathway-specific manner, but surprisingly, with different kinetics, as the genes derived from one of the parent species (the non-Saccharomyces cerevisiae-like) are alleviated from repression much faster than the genes from the other parent (the S. cerevisiae-like). This probably reflects differing physiological adaptation of the parent species, and the finding may contribute to the general understanding of hybrid speciation.

Expression of CC chemokine receptor-7 and regional lymph node metastasis of B16 murine melanoma.

CC chemokine receptor-7 (CCR7), which plays a critical role in the migration of activated dendritic cells to regional lymph nodes via afferent lymphatic vessels, is also expressed by human breast and melanoma cell lines. Because neoplastic cells also enter lymphatic vessels before metastasis to the lymph nodes, we investigated whether CCR7 expression enhances metastasis of B16 murine melanoma cells to regional lymph nodes. B16 cells were transduced with a retroviral vector containing CCR7 complementary DNA (CCR7-B16 cells) or with vector alone (pLNCX2-B16 control cells). The functional assay for CCR7 protein was Ca(2+) flux stimulated by the chemokine CCL21, a CCR7-specific ligand produced by lymphatic endothelial cells. B16 tumor cells were injected into the footpad of mice. Tumor cell metastasis to draining lymph nodes was assessed by measuring messenger RNA (mRNA) for tyrosinase-related protein-1 (TRP), a melanocyte-specific enzyme, with real-time, quantitative reverse transcription-coupled polymerase chain reaction. All statistical tests were two-sided. One week after injection into the footpad, 701-fold (95% confidence interval [CI] = 64- to 1336-fold) more TRP mRNA was detected in draining lymph nodes from CCR7-B16 cell-injected mice than in those from control cell-injected mice. Three weeks after footpad injection, 58% (11 of 19) of the draining lymph nodes from CCR7-B16 cell-injected mice and 5% (one of 19) of those from control mice showed gross metastases (P<.001). CCR7-B16 cells isolated from lymph node metastases retained functional CCR7 expression. Lymph node metastasis of CCR7-B16 cells was blocked by neutralizing anti-CCL21 antibodies (metastasis in none of five lymph nodes) but not by control immunoglobulin G (three of five). Enhanced metastasis of CCR7-B16 cells was specific for a lymphatic route because both CCR7-B16 and control cells co-injected intravenously metastasized to the lung at the same frequency. Expression of a single chemokine receptor gene, CCR7, increased B16 cell metastasis to draining lymph nodes, suggesting that cancer cells may co-opt normal mechanisms of lymph node homing during metastasis.

Molecular Cloning of a Third Member of the Potassium-dependent Sodium-Calcium Exchanger Gene Family,NCKX3

We describe here the identification and characterization of a novel member of the family of K(+)-dependent Na(+)/Ca(2+) exchangers, NCKX3 (gene SLC24A3). Human NCKX3 encodes a protein of 644 amino acids that displayed a high level of sequence identity to the other family members, rod NCKX1 and cone/neuronal NCKX2, in the hydrophobic regions surrounding the "alpha -repeat" sequences thought to form the ion-binding pocket for transport. Outside of these regions NCKX3 showed no significant identity to other known proteins. As anticipated from this sequence similarity, NCKX3 displayed K(+)-dependent Na(+)/Ca(2+) exchanger activity when assayed in heterologous expression systems, using digital imaging of fura-2 fluorescence, electrophysiology, or radioactive (45)Ca(2+) uptake. The N-terminal region of NCKX3, although not essential for expression, increased functional activity at least 10-fold and may represent a cleavable signal sequence. NCKX3 transcripts were most abundant in brain, with highest levels found in selected thalamic nuclei, in hippocampal CA1 neurons, and in layer IV of the cerebral cortex. Many other tissues also expressed NCKX3 at lower levels, especially aorta, uterus, and intestine, which are rich in smooth muscle. The discovery of NCKX3 thus expands the K(+)-dependent Na(+)/Ca(2+) exchanger family and suggests this class of transporter has a more widespread role in cellular Ca(2+) handling than previously appreciated.

Prediction of the coding sequences of unidentified human genes. XXI. The complete sequences of 60 new cDNA clones from brain which code for large proteins.

As an extension of a sequencing project of human cDNA clones which encode large proteins of unidentified genes, we herein present the entire sequences of 60 cDNA clones for the genes named KIAA1879-KIAA1938. The cDNA clones were isolated from size-fractionated cDNA libraries derived from human fetal brain, adult whole brain and amygdala, and their protein-coding sequences were predicted. Thirty-seven cDNA clones entirely sequenced in this study were selected as cDNAs which have coding potentiality by in vitro transcription/translation experiments, and the remaining 23 cDNA clones were chosen by computer-assisted analysis of terminal sequences of cDNAs. The average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here were 4.5 kb and 2.2 kb (733 amino acid residues), respectively. Sequence analyses against the public databases enabled us to annotate the functions of the predicted products of the 25 genes; 84% of these predicted gene products (21 gene products) were classified into proteins related to cell signaling/communication, nucleic acid management, and cell structure/motility. In addition to the sequence information about these 60 genes, their expression profiles were also studied in some human tissues including brain regions by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Prediction of the Coding Sequences of Unidentified Human Genes. XX. The Complete Sequences of 100 New cDNA Clones from Brain Which Code for Large Proteins in vitro

To accumulate information on the coding sequences of unidentified genes, we have carried out a sequencing project of human cDNA clones which encode large proteins. We herein present the entire sequences of 100 cDNA clones of unidentified human genes, named KIAA1776 and KIAA1780-KIAA1878, from size-fractionated cDNA libraries derived from human fetal brain, adult whole brain, hippocampus and amygdala. Most of the cDNA clones to be entirely sequenced were selected as cDNAs which were shown to have coding potentiality by in vitro transcription/translation experiments, and some clones were chosen by using computer-assisted analysis of terminal sequences of cDNAs. Three of these clones (fibrillin2/KIAA1776, MEGF10/KIAA1780 and MEGF11/KIAA1781) were isolated as genes encoding proteins with multiple EGF-like domains by motif-trap screening. The average sizes of the inserts and corresponding open reading frames of eDNA clones analyzed here reached 4.7 kb and 2.4 kb (785 amino acid residues), respectively. From the results of homology and motif searches against the public databases, the functional categories of the predicted gene products of 54 genes were determined; 93% of these predicted gene products (50 gene products) were classified as proteins related to cell signaling/communication, nucleic acid management, or cell structure/motility. To collect additional information on these genes, their expression profiles were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Prediction of the coding sequences of unidentified human genes. XXII. The complete sequences of 50 new cDNA clones which code for large proteins.

As an extension of human cDNA projects for accumulating sequence information on the coding sequences of unidentified genes, we herein present the entire sequences of 50 cDNA clones, named KIAA1939-KIAA1988. cDNA clones to be entirely sequenced were selected by two approaches based on their protein-coding potentialities prior to sequencing: 10 cDNA clones were chosen because their encoding proteins had a molecular mass larger than 50 kDa in an in vitro transcription/translation system; the remaining 40 cDNA clones were selected because their putative proteins-as determined by analysis of the genomic sequences flanked by both the terminal sequences of cDNAs using the GENSCAN gene prediction program-were larger than 400 amino acid residues. According to the sequence data, the average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here were 4.6 kb and 1.9 kb (643 amino acid residues), respectively. From the results of homology and motif searches against the public databases, the functional categories of the 31 predicted gene products could be assigned; 25 of these predicted gene products (81%) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility. The expression profiles of the genes were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, the products of which were quantified by enzyme-linked immunosorbent assay.

Elevated transferrin concentration in cerebral spinal fluid after subarachnoid hemorrhage

Calcium-elevating protein cross-reacted with anti-human transferrin (TF) was purified in cerebrospinal fluid (CSF) after subarachnoid hemorrhage (SAH). The concentration of TF in CSF after SAH was measured, and the effects of TF on cultured smooth muscle cells (SMCs) were evaluated in order to understand the relationship between TF and cerebral vasospasm. Cisternal CSF samples were collected from 12 patients (seven men and 13 women) with SAH due to the rupture of a cerebral aneurysm, and eight control subjects. The patients were divided into two groups: (1) those presenting no clinical and radiological evidence of vasospasm (the non-vasospasm group; three men and 10 women, mean age 54.7 years), and (2) those presenting evidence of vasospasm (the vasospasm group; four men and three women, mean age 58.3 years). The concentration of TF in CSF was measured using Speriol micro-transferrin measure assay method. Nitrite accumulation in the culture media of SMCs incubated with TF was determined by diazotization method. The mRNA levels of inducible isoform of NOS (iNOS) in SMC incubated with TF were measured by the reverse transcription-coupled polymerase chain reaction method. Levels of TF were markedly different in the SAH and the control subjects. In the control group, all subjects had no detectable quantity of TF. In contrast, all patients after SAH had quantifiable TF in their CSF. Moreover, there was a significant difference between the vasospasm group and the non-vasospasm group in TF levels (p < 0.05). In the vasospasm group, the average value was 10.43±2.8 mg dl-1 In the non-vasospasm group, the average was 3.69±0.31 mg dl-1. The nitrite content in the culture medium incubated with TF was three times the content of control. TF also induced iNOS mRNA in SMC. This study demonstrated that an elevation of TF concentration in CSF after SAH was detected and iNOS mRNA in SMCs was also induced by TF. This may be involved in some roles of the development of the pathological series of events after SAH, including vasospasm. [Neurol Res 2000; 22: 797-801]

Sequence analysis, gene expression, and chromosomal assignment of mouse Borg4 gene and its human orthologue.

The Borg (binder of Rho GTPases) family proteins interact with CDC42 and TC10 in a guanosine triphosphate (GTP)-dependent manner. We have isolated a full-length cDNA of the mouse Borg4 gene, which is a member of this family. Sequence analysis revealed that this gene encoded a putative 349-amino acid protein. By reverse transcription--coupled polymerase chain reaction (RT-PCR) analysis, we observed that Borg4 was expressed ubiquitously in adult tissues. Additionally, we determined the entire cDNA sequence of the putative human Borg4 orthologue. By fluorescence in situ hybridization, mouse Borg4 and the putative human orthologue have been assigned to mouse chromosome 11E and human chromosome 17q24-25, which has been described as syntenic to the mouse region.

CDNA cloning of a human RAB26-related gene encoding a Ras-like GTP-binding protein on chromosome 16p13.3 region.

Members of the RAB protein family are important regulators of vesicular fusion and trafficking. A putative new member of the RAB family of genes was identified through a public database search, and its full-length cDNA was isolated from a human fetal brain cDNA library. The predicted protein product of the gene consists of 190 amino acid residues and has 87% identity with rat Rab26. Thus, we designated this gene as the human RAB26-related gene. Reverse transcription-coupled polymerase chain reaction (RT-PCR) demonstrated that the RAB26-related messenger RNA was predominantly expressed in adult and fetal brain. Furthermore, an RT-PCR experiment for brain subregions showed that the mRNA was highly expressed in the amygdala, cerebellum, caudate nucleus, and hippocampus. By PCR-based analysis with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid panel, the gene was mapped to the chromosome 16p13.3 region between markers WI-7742 and WI-3061. The RAB26-related gene consists of eight exons that span about 44kb of the genome DNA.

Differential regulation of adult and embryonic glutamate decarboxylases in rat dentate granule cells after kainate-induced limbic seizures

In adult brain, the inhibitory GABAergic neurons utilize two distinct molecular forms of the GABA-synthesizing enzyme glutamate decarboxylase (GAD), GAD65 and GAD67. During embryonic development, two truncated forms of GAD67 are also expressed (GAD25 and GAD44), which are translated from two embryonic-specific splice variants of GAD67 messenger RNA. It has recently been established that the excitatory dentate granule cells, in addition to the neurotransmitter glutamate, also contain low levels of GABA and GAD67, which are increased after limbic seizures. To study the seizure-induced activation of glutamate decarboxylase, we investigated the expression of both embryonic and adult glutamate decarboxylase messenger RNAs in the adult rat hippocampus after kainic acid administration by semi-quantitative reverse transcription-coupled polymerase chain reaction, in situ hybridization and immunoblotting. We observed a rapid induction of the embryonic glutamate decarboxylase messenger RNA in the granule cells of dentate gyrus. The expression of embryonic glutamate decarboxylase transcripts, identified here as the splice variant that contains exon 7/B, peaked at about 2h after kainic acid injection and gradually returned to nearly basal levels by 24h. Strikingly, this transient induction of embryonic glutamate decarboxylase messenger RNA was not accompanied by concomitant synthesis of its corresponding protein product GAD25. In contrast, the adult GAD67 messenger RNA and protein were both clearly up-regulated in granule cells, albeit with a certain delay, reaching a maximum around 4–6h after kainic acid injection and gradually returned to control levels by 24h. GAD65 remained unchanged at both messenger RNA and protein levels during the studied period. These characteristic and highly reproducible changes in the synthesis of glutamate decarboxylases indicate that GAD67 is the predominant form of glutamate decarboxylases involved in the elevated synthesis of GABA during seizures and suggest that the transient induction of the embryonic GAD67 messenger RNA that contains exon 7/B, but not GAD25 protein, may exert a role solely in the subsequent up-regulation of adult GAD67 transcription.Expression of the messenger RNA encoding for an alternatively spliced, truncated form of the GABA-synthesizing enzyme glutamate decarboxylase was detected in dentate granule cells briefly after kainic acid-induced seizures. Just as during embryonic development, expression of the alternatively spliced messenger RNA was transient and followed by transcription of its adult form, indicating a possible recapitulation of an embryonic program of gene expression in adult granule cells after epileptic seizures.

Isolation of Novel Mouse Genes Differentially Expressed in Brain Using cDNA Microarray

The major issue in the post-genome sequencing era is determination of gene expression patterns in variety of biological systems. A microarray system is a powerful technology for analyzing the expression profile of thousands of genes at one experiment. In this study, we identified highly expressed genes in mouse brain using the cDNA microarray carrying 2304 cDNAs derived from oligo-capped mouse cDNA library. Nine genes were highly expressed in adult mouse brain compared to kidney, liver, and skeletal muscle. Tissue distribution analysis by reverse transcription-coupled polymerase chain reaction (RT-PCR) revealed that consistent with the microarray data, all of the selected 9 genes were predominantly expressed in the brain. A database search showed that 5 of the 9 genes, MBP, SC1, HiAT3, S100 protein-beta, and SNAP25, were previously known to be expressed at high level in the brain and in the nervous system. One gene was highly sequenced similar to rat S-Rex-s/human NSP-C, suggesting that the gene is a mouse homologue. The remaining three genes did not match to known genes in the GenBank/EMBL database, indicating that these are novel genes highly expressed in the brain. Taken together, our cDNA microarray system can be an excellent tool for identifying differentially expressed genes in mouse brain.

Regulation of nitric oxide synthase to promote cytostasis in ovarian follicular development.

Our own recent studies have demonstrated that inducible nitric oxide synthase (iNOS) is predominantly localized in granulosa cells of healthy immature follicles in the rat ovary, whereas granulosa cells of either healthy mature follicles or follicles destined to be atretic are devoid of iNOS. These findings suggest that iNOS is pivotal for immature follicles to remain dormant. To test this hypothesis, we examined the effects of a GnRH agonist (buserelin), a proapoptotic substance, and epidermal growth factor (EGF), a mitogenic and, consequently, antiapoptotic factor, on the amount of iNOS mRNA in rat granulosa cells. Administration of buserelin in immature female rats transiently diminished iNOS mRNA levels in the ovaries as determined by Northern blot analysis. In cultured rat granulosa cells, buserelin and EGF increased the incidence of apoptosis and DNA synthesis, respectively, whereas both reduced iNOS mRNA levels as determined by reverse transcription-coupled polymerase chain reaction. The concomitant addition of S-nitroso-N-acetyl-DL-penicillamine, an NO donor, together with buserelin or EGF eliminated the observed effects of these substances (i.e., induction of apoptosis and stimulation of DNA synthesis, respectively). These results suggest that the changes in developmental status of immature follicles either into development or atresia are associated with reduced iNOS levels in granulosa cells, thus reinforcing the notion of NO as a cytostatic factor in ovarian follicles.

Distribution of leukotriene B4 receptors in human hematopoietic cells

Leukotriene B4 (LTB4), a product of arachidonic acid metabolism, plays an important role in inflammatory responses. We have cloned from human erythroleukemia cells, a G protein-coupled receptor, designated P2Y7, which was later identified as the receptor for LTB4 (B-LTR). We have investigated the distribution of LTB4 receptors in various hematopoietic cells. Northern blotting and reverse transcription-coupled polymerase chain reaction (RT-PCR) analyses using radiolabeled LTB4 receptor cDNA as a probe indicated the presence of LTB4 receptor mRNA in peripheral blood leukocytes but not in platelets. Flow cytometry analysis of peripheral blood cells using specific LTB4 receptor antibodies revealed that monocytes, granulocytes, and lymphocytes, but not platelets, express LTB4 receptors. RT-PCR-Southern hybridization analysis revealed that peripheral blood leukocytes and human umbilical vein endothelial cells express the LTB4 receptor. Of the hematopoietic cell lines tested, promonocytic U937 cells, promyelocytic HL-60 cells, K562 cells, and human erythroleukemia cells express the LTB4 receptor. These results suggest a physiological role for the LTB4 receptor in the stimulation of monocytes, neutrophils, and endothelial cells.

Effects of AMP derivatives on cyclic AMP levels in NG108-15 cells.

1. In NG108-15 neuroblastomaxglioma hybrid cells, ATP stimulates intracellular cyclic AMP formation, which is inhibited by both adenosine (P(1)) and P2 receptor antagonists. In the present study, we examined the effects of several AMP derivatives in NG108-15 cells and mouse neuroblastoma N18TG-2 cells. 2. Adenosine 2'-monophosphate (A2P), adenosine 3'-monophosphate (A3P) and adenosine 5'-phosphosulphate (A5PS) increased cyclic AMP levels with similar concentration-dependencies in NG108-15 cells. 3. Increases in cyclic AMP by AMP derivatives were inhibited by the P2 receptor antagonist PPADS, but not by suramin. Effects of AMP derivatives were also inhibited by P(1) receptor antagonists ZM241385, XAC, DPCPX and partially by alloxazine. The ecto-nucleotidase inhibitor alpha, beta-methyleneADP was without effect. 4. In contrast, AMP derivatives did not change cyclic AMP levels in N18TG-2 cells. Accumulation of cyclic AMP in N18TG-2 cells was stimulated by adenosine A(2) receptor agonists CGS21680 and NECA, but not by ATP or beta, gamma-methyleneATP, agonists for cyclic AMP production in NG108-15 cells. 5. Reverse transcription-coupled polymerase chain reaction (RT - PCR) analyses revealed that N18TG-2 cells express both A(2A) and A(2B) receptors, while NG108-15 cells express mainly A(2A) receptors. 6. AMP derivatives did not affect the P2X and P2Y receptors expressed in NG108-15 cells. 7. These results suggest that A2P, A3P and A5PS act as agonists for cyclic AMP production and that these compounds are valuable tools for determinating the mechanism of ATP-stimulated cyclic AMP response in NG108-15 cells.

A non-stop antisense reading frame in the grp78 gene of Neurospora crassa is homologous to the Achlya klebsiana NAD-gdh gene but is not being transcribed

A long non-stop reading frame exists on the antisense strand of the grp78 gene (cDNA and genomic DNA) of Neurospora crassa. Computer analysis revealed a strong similarity of the putative antisense protein to the 10th exon of the NAD-dependent glutamate dehydrogenase gene ( NAD-gdh) of Achlya klebsiana, which is itself located on the complementary strand of a transcribed hsc70 gene homologue. In Neurospora, no grp78 antisense mRNA was detected by Northern blot and reverse transcription-coupled polymerase chain reaction analyses, indicating that this long reading frame is not being transcribed. Hypotheses for the presence of such unexpressed non-stop reading frames are discussed.

A non-stop antisense reading frame in the grp78 gene of Neurospora crassa is homologous to the Achlya klebsiana NAD-gdh gene but is not being transcribed.

A long non-stop reading frame exists on the antisense strand of the grp78 gene (cDNA and genomic DNA) of Neurospora crassa. Computer analysis revealed a strong similarity of the putative antisense protein to the 10th exon of the NAD-dependent glutamate dehydrogenase gene (NAD-gdh) of Achlya klebsiana, which is itself located on the complementary strand of a transcribed hsc70 gene homologue. In Neurospora, no grp78 antisense mRNA was detected by Northern blot and reverse transcription-coupled polymerase chain reaction analyses, indicating that this long reading frame is not being transcribed. Hypotheses for the presence of such unexpressed non-stop reading frames are discussed.