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Results for “Nuclease Protection Assays”

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PubMed1993

GABA transporter mRNA: in vitro expression and quantitation in neonatal rat and postmortem human brain.

A previously isolated rat cDNA clone encoding the membrane transporter for the neurotransmitter gamma-aminobutyric acid was expressed in transfected COS cells. The resultant transporter protein was characterized kinetically and pharmacologically. The apparent Kt (6.1 microM) and the pharmacological profile of a neuronal-type transporter observed in these mammalian cells were consistent with previous data obtained in Xenopus laevis oocytes. Post-natal levels of gamma-aminobutyric acid transporter mRNA in rat cerebellum, cerebral cortex and striatum (as measured by nuclease protection assay) transiently exceeded levels present in the adult brain. Human gamma-aminobutyric acid transporter mRNA also was measured by nuclease protection assay using as probe a human transporter cDNA homolog obtained by polymerase chain reaction. These studies suggest that quantitation of rat and human gamma-aminobutyric acid transporter mRNAs may provide a useful index of transporter gene expression.

Adult↗

PubMed2002

Expression of peroxisome proliferator-activated receptors alpha and gamma in differentiating human colon carcinoma Caco-2 cells.

The expression of peroxisome proliferator-activated receptors alpha (PPARalpha) and gamma (PPARgamma) was studied in the human adenocarcinoma Caco-2 cells induced to differentiate by long term culture (15 days). The differentiation of Caco-2 cells was attested by increases in the activities of sucrase-isomaltase and alkaline phosphatase (two brush border enzymes), fatty acyl-CoA oxidase (AOX) and catalase (two peroxisomal enzymes), by an elevation in the protein levels of villin (a brush border molecular marker), AOX, peroxisomal bifunctional enzyme (PBE), catalase and peroxisomal membrane protein of 70 kDa (PMP70). and by the appearance of peroxisomes. The expression of PPARalpha and PPARgamma was investigated by Western blotting, immunocytochemistry, Northern blotting and S1 nuclease protection assay during the differentiation of Caco-2 cells. The protein levels of PPARalpha, PPARgamma, and PPARgamma2 increased gradually during the time-course of Caco-2 cell differentiation. Immunocytochemistry revealed that PPARalpha and gamma were localized in cell nuclei. The PPARgamma1 protein was encoded by PPARgamma3 mRNA because no signal was obtained for PPARgamma1 mRNA using a specific probe in S1 nuclease protection assay. The amount of PPARgamma3 mRNA increased concomitantly to the resulting PPARgamma1 protein. On the other hand, the mRNA of PPARalpha and PPARgamma2 were not significantly changed, suggesting that the increase in their respective protein was due to an elevation of the translational rate. The role played by the PPAR subtypes in Caco-2 cell differentiation is discussed.

Blotting, Northern↗

PubMed1995

Expression and regulation of rat 6-pyruvoyl tetrahydropterin synthase mRNA.

6-Pyruvoyl tetrahydropterin synthase catalyzes the second step in the biosynthesis of tetrahydrobiopterin. In the present study, the reverse transcription-polymerase chain reaction technique was used to clone a portion of 6-pyruvoyl tetrahydropterin synthase cDNA from rat pineal gland RNA. The sequence of this cDNA was found to be essentially identical to that previously reported for the rat liver. 6-Pyruvoyl tetrahydropterin synthase mRNA levels in various rat tissues, including the brain, were then analyzed by Northern blot and nuclease protection assay. A single 1.35 kb transcript of 6-pyruvoyl tetrahydropterin synthase mRNA was detected by Northern blot analysis in the rat adrenal gland, brain-stem, and liver. Quantitation by nuclease protection assay demonstrated that 6-pyruvoyl tetrahydropterin synthase mRNA was most abundant in the adrenal gland, kidney, and pineal gland (19.5-25.5 amol/microgram RNA). Relatively homogeneous levels of 6-pyruvoyl tetrahydropterin synthase mRNA were found in various brain regions including the cerebellum, substantia nigra and locus coeruleus (4.12-12 amol/microgram RNA). In the adrenal gland, 6-pyruvoyl tetrahydropterin synthase and tyrosine hydroxylase mRNAs were elevated between 3 and 4-fold 24 h after a single dose of reserpine (10 mg/kg), a treatment known to increase tetrahydrobiopterin levels in this tissue. This result suggests that although 6-pyruvoyl tetrahydropterin synthase is not believed to be rate-limiting in the tetrahydrobiopterin biosynthetic pathway, control of gene expression for this enzyme may play an essential role in regulating the synthesis of this important cofactor.

5'-Nucleotidase↗

PubMed1997

Molecular evolution and functional relevance of the chalcone synthase genes of pea.

We have isolated seven genomic chalcone synthase (CHS) genes and six classes of CHS cDNA from elicitor-treated pea tissues. Comparison of the nucleotide sequences of the coding regions revealed the existence of eight members of the CHS gene family in pea. These can essentially be divided into three groups (PSCHS1, 2 and 8; PSCHS3, 4 and 5; and PSCHS6 and 7) on the basis of nucleotide and or amino acid sequence comparisons of the coding regions, introns and promoter regions. We previously reported that the accumulation of CHS mRNAs is induced by elicitor treatment. Accumulation of CHS mRNA was observed mainly in roots and very little was found in floral organs. To specifically detect expression of each CHS gene in various types of pea cells. S1 nuclease protection assays were performed. Interestingly, the classification of the eight members of the CHS gene family based on the sequence identity was found to reflect their expression patterns as determined by the S1 nuclease protection assay. The first group of CHS genes, PSCHS1, 2 and 8, was strongly induced not only by elicitor treatment and UV irradiation but is also constitutively expressed in root and flower tissues. The second group, PSCHS3, 4 and 5, was also strongly induced by elicitor treatment and UV irradiation but is constitutively expressed only in root. Expression of the third group, PSCHS6 and 7 was barely detectable in any of the organs tested and was not influenced by environmental stimuli such as elicitor or UV. Furthermore, sequence analysis of the promoter region of each member of the CHS gene family revealed that putative cis-regulatory elements, such as Box-I. Box-II and G-Box, were conserved only in PSCHS1, 2, 3, 4 and 5. From these results we propose that an ancestral CHS gene might have given rise to defense response-related (UV irradiation- and elicitor-responsive) and -unrelated (unresponsive) genes at an early stage of evolution, followed by divergence within these subclasses based upon the developmental program in pea.

Acyltransferases↗

PubMed1998

Tissue distribution of rat glucagon receptor and GLP-1 receptor gene expression.

The regulation of glucose metabolism by glucagon and GLP-1 is well established, but novel functions for these and other proglucagon-derived peptides are less well defined. This paper highlights the diversity of both GLP-1 and glucagon activity by studying the tissue distribution of glucagon and GLP-1 receptor gene expression by both Southern blot analysis of RT-PCR products and nuclease protection assays. By Southern blot analysis of RT-PCR products, GLP-1 receptor mRNA was detected in lung, hypothalamus, hippocampus, cerebral cortex, kidney, pancreas, and throughout the gastrointestinal tract. Glucagon receptor expression was detected in liver, kidney, spleen, thymus, adrenal glands, pancreas, cerebral cortex, lung, and throughout the gastrointestinal tract. Nuclease protection assay revealed glucagon receptor expression to be highest in liver and kidney, whereas GLP-1 receptor expression was only detected by protection assay in lung, stomach, and large bowel. Despite previous evidence that other receptors for proglucagon-derived peptides may exist, no evidence of novel receptors or multiple isoforms of the glucagon and GLP-1 receptors was found, indicating that the two cloned receptors may mediate all the effects of proglucagon-derived peptides, or that novel receptors may share less homology with the glucagon and GLP-1 receptors than previously anticipated.

Animals↗

PubMed2005

Expression of the alpha7 nicotinic acetylcholine receptor in human lung cells.

BACKGROUND: We and others have shown that one of the mechanisms of growth regulation of small cell lung cancer cell lines and cultured pulmonary neuroendocrine cells is by the binding of agonists to the alpha7 neuronal nicotinic acetylcholine receptor. In addition, we have shown that the nicotine-derived carcinogenic nitrosamine, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a high affinity agonist for the alpha7 nicotinic acetylcholine receptor. In the present study, our goal was to determine the extent of alpha7 mRNA and protein expression in the human lung. METHODS: Experiments were done using reverse transcription polymerase chain reaction (RT-PCR), a nuclease protection assay and western blotting using membrane proteins. RESULTS: We detected mRNA for the neuronal nicotinic acetylcholine receptor alpha7 receptor in seven small cell lung cancer (SCLC) cell lines, in two pulmonary adenocarcinoma cell lines, in cultured normal human small airway epithelial cells (SAEC), one carcinoid cell line, three squamous cell lines and tissue samples from nine patients with various types of lung cancer. A nuclease protection assay showed prominent levels of alpha7 in the NCI-H82 SCLC cell line while alpha7 was not detected in SAEC, suggesting that alpha7 mRNA levels may be higher in SCLC compared to normal cells. Using a specific antibody to the alpha7 nicotinic receptor, protein expression of alpha7 was determined. All SCLC cell lines except NCI-H187 expressed protein for the alpha7 receptor. In the non-SCLC cells and normal cells that express the alpha7 nAChR mRNA, only in SAEC, A549 and NCI-H226 was expression of the alpha7 nicotinic receptor protein shown. When NCI-H69 SCLC cell line was exposed to 100 pm NNK, protein expression of the alpha7 receptor was increased at 60 and 150 min. CONCLUSION: Expression of mRNA for the neuronal nicotinic acetylcholine receptor alpha7 seems to be ubiquitously expressed in all human lung cancer cell lines tested (except for NCI-H441) as well as normal lung cells. The alpha7 nicotinic receptor protein is expressed in fewer cell lines, and the tobacco carcinogen NNK increases alpha7 nicotinic receptor protein levels.

Biomarkers, Tumor↗

PubMed1999

Activation of the transforming growth factor beta signaling pathway and induction of cytostasis and apoptosis in mammary carcinomas treated with the anticancer agent perillyl alcohol.

The mechanisms of action of the anticancer agent perillyl alcohol (POH), presently in Phase II clinical trials, were investigated in advanced rat mammary carcinomas. Gross and ultrastructural morphology of POH-mediated tumor regression indicated that apoptosis accounted for the marked reduction in the epithelial compartment. Characterization of cell growth and death indices revealed that apoptosis was induced within 48 h of chemotherapy, before the induction of cytostasis. RNA expression studies, based on a multiplexed-nuclease protection assay, demonstrated that cell cycle- and apoptosis-related genes were differentially expressed within 48 h of POH treatment; p21(Cip1/WAF1), bax, bad, and annexin I were induced; cyclin E and cyclin-dependent kinase 2 were repressed; and bcl-2 and p53 were unchanged. Next, a potential role for transforming growth factor beta (TGF-beta) signaling in POH-mediated carcinoma regression was explored. RNA expression studies, again based on a multiplexed-nuclease protection assay, showed that TGF-beta-related genes were induced and temporally regulated during POH treatment: (a) c-jun and c-fos were transiently induced within 12 h of chemotherapy; (b) TGF-beta1 was induced within 24 h of chemotherapy; (c) the mannose 6-phosphate/insulin-like growth factor II receptor and the TGF-beta type I and II receptors were induced within 48 h of chemotherapy; and (d) smad3 was induced during active carcinoma regression. In situ protein expression studies, based on fluorescence-immunohistochemistry in concert with confocal microscopy, confirmed up-regulation and demonstrated colocalization of TGF-beta1, the mannose 6-phosphate/insulin-like growth factor II receptor, the TGF-beta type I and II receptors, and Smad2/Smad3 in epithelial cells. Nuclear localization of Smad2/Smad3 indicated that the TGF-beta signaling pathway was activated in regressing carcinomas. Subpopulations of Smad2/Smad3-positive and apoptotic nuclei colocalized, indicating a role for Smads in apoptosis. Thus, Smads may serve as a potential biomarker for anticancer activity. Importantly, none of the POH-mediated anticancer activities were observed in normal mammary gland.

Animals↗

PubMed1997

B-cell lines immortalized with an Epstein-Barr virus mutant lacking the Cp EBNA2 enhancer are biased toward utilization of the oriP-proximal EBNA gene promoter Wp1.

During Epstein-Barr virus (EBV) latent infection of B lymphocytes in vitro, six viral nuclear antigens (EBNAs) are expressed from one of two promoters, Cp or Wp, whose activities have previously been shown to be mutually exclusive in established lymphoblastoid cell lines. Initially after infection, the EBNA genes are transcribed from Wp, which is present in multiples copies within the major internal repeat of EBV. Approximately 48 to 72 h postinfection, Wp is downregulated, with a corresponding increase in transcription from Cp. An EBNA2-responsive enhancer exists upstream of Cp, and a role for EBNA2 in the induction of Cp activity during the establishment of viral latency has previously been proposed (Woisetschlaeger et al., Proc. Natl. Acad. Sci. USA 87:1725-1729, 1991). To critically assess the potential role for this enhancer region in determining relative usage of Cp and Wp, an EBNA2 enhancer deletion mutant virus was generated. Lymphoblastoid cell lines were screened by PCR and Southern blotting for the presence of mutant virus harboring the EBNA2 enhancer deletion. A quantitative S1 nuclease protection assay was developed to allow comparison of relative Cp and Wp activities for the cell lines containing mutant virus and those of the wild-type recombinants which lacked the enhancer deletion. In general, the wild-type recombinants had higher levels of Cp-initiated transcripts than Wp-initiated transcripts. In contrast, the Cp EBNA2 enhancer deletion mutants exhibited a strong bias toward Wp activity. Notably, only the first Wp (oriP-proximal Wp; Wp1) appears active in these mutants. S1 nuclease protection assays using a probe which hybridizes to the W2 exon, contained in both Cp- and Wp-initiated transcripts, indicated that the total level of transcription from Cp and Wp remained the same in wild-type and EBNA2 enhancer mutant cell lines. The presence of both Cp and Wp activity in the wild-type recombinants, as well as in newly derived lymphoblastoid cell lines established with the prototype B95.8 virus, demonstrated that Cp and Wp activities are not always mutually exclusive.

B-Lymphocytes↗

PubMed2001

Differential regulation of ftsZ transcription during septation of Streptomyces griseus.

Streptomyces has been known to form two types of septa. The data in this research demonstrated that Streptomyces griseus forms another type of septum near the base of sporogenic hyphae (basal septum). To understand the regulation of the septation machinery in S. griseus, we investigated the expression of the ftsZ gene. S1 nuclease protection assays revealed that four ftsZ transcripts were differentially expressed during morphological differentiation. The vegetative transcript (emanating from P(veg)) is present at a moderate level during vegetative growth, but is switched off within the first 2 h of sporulation. Two sporulation-specific transcripts predominantly accumulated, and the levels increased by approximately fivefold together shortly before sporulation septa begin to form. Consistently, the sporulation-specific transcripts were expressed much earlier and more abundantly in a group of nonsporulating mutants that form their sporulation septa prematurely. Promoter-probe studies with two different reporter systems confirmed the activities of the putative promoters identified from the 5' end point of the transcripts. The levels and expression timing of promoter activities were consistent with the results of nuclease protection assays. The aseptate phenotype of the P(spo) mutant indicated that the increased transcription from P(spo) is required for sporulation septation, but not for vegetative or basal septum formation.

Bacterial Proteins↗

PubMed1991

Testicular inhibition of tyrosine hydroxylase expression in tuberoinfundibular and nigrostriatal dopaminergic neurons.

We have investigated, in young and aged rats, tuberoinfundibular dopaminergic (TIDA) and nigrostriatal neurons of the brain, and the role of the testes and anterior pituitary (AP) on 1) the in situ activity of tyrosine hydroxylase (TH), 2) translation of TH mRNA, as reflected by the mass of TH, and 3) transcription of the TH gene, as revealed by the mass of TH mRNA. The median eminence (ME) and corpus striatum (CS) were used as the sources of proximal neurites of the TIDA neurons and nigrostriatal neurons, respectively. The arcuate-periventricular nuclei of the ventral hypothalamus were used as the source of perikarya of the TIDA neurons, and substantia nigra (SN) nuclei of the midbrain were used as the source of perikarya of nigrostriatal neurons. The in situ activity of TH was calculated using the rate of accumulation of dihydroxyphenylalanine after pharmacological inhibition of dihydroxyphenylalanine decarboxylase activity. TH mass and TH mRNA were measured using an immunoblot assay and an S1 nuclease protection assay, respectively. Compared to intact animals, orchidectomized young and aged male rats had significantly (P less than 0.001) increased in situ activity of TH in the ME and CS, but not the SN. Orchidectomy also caused a significant (P less than 0.01) increase in the quantity of TH in the ME and a 2- to 3-fold increase in TH in the CS and SN. In contrast to castration, AP grafts, implanted in the lateral ventricles of the brain, caused a significant (P less than 0.001) increase only in TH activity in the ME. No effect of AP grafts was seen on TH activity of the CS or SN. AP grafts had no effect on the amount of TH in the ME, CS, or SN. In the TIDA neurons of young males and in the nigrostriatal neurons of young and aged castrates, the amount of TH mRNA was not different from that in intact males. AP grafts had no effect on TH mRNA in these dopaminergic neurons. These studies show that TIDA neurons and nigrostriatal neurons share some common aspects in their regulation and are dissimilar in others. AP grafts stimulate TH activity in young as well as aged TIDA neurons, but not in nigrostriatal neurons. Castration leads to an increase in TH mass in both TIDA and nigrostriatal neurons. The increase in TH mass is not associated with an increase in the amount of TH mRNA. It is suggested that castration results in increased translation of TH mRNA, but not transcription of the TH gene.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗

PubMed1983

Analysis of rabbit beta-like globin gene transcripts during development.

We have analyzed the differential expression of a family of beta-like globin genes during the development of rabbits, from four days post implantation to one week before birth. The family is composed of four genes, arranged 5'-beta 4-beta 3-psi beta 2-beta 1-3' on the chromosome; psi beta 2 is an inactive pseudogene. Using the technique of hybrid-arrested translation in vitro, we have identified the embryo-specific globin polypeptides encoded by genes beta 3 and beta 4. The beta 3 and beta 4 globins are replaced by the adult beta 1 globin halfway through gestation; this corresponds temporally with the switch in site of erythropoiesis from the embryonic yolk sac to the fetal liver. The decline in production of beta 3 globin polypeptide precedes the decline in beta 4 globin. Transcripts from genes beta 1, beta 3 and beta 4 were analyzed at progressive stages of gestation by a blot-hybridization assay and by an S1 nuclease protection assay. Mature messenger RNA and presumptive precursor RNAs from genes beta 3 and beta 4 are synthesized abundantly in embryonic erythroid cells but only at very low levels later in fetal development. Conversely, precursor and mature mRNA from gene beta 1 are found at very low levels in embryos but are abundant in fetal and adult erythroid cells. The co-ordinate appearance of precursor RNA, mRNA and polypeptide from all three active genes indicates that the primary developmental regulation of this gene family is exerted at the level of transcription. RNA species larger than the expected precursors were observed when the RNA was denatured with formaldehyde but not when methylmercury was the denaturant. These large RNAs are a formaldehyde-generated artifact, possibly a result of cross-linking globin transcripts to ribosomal RNA. We observe no extensive stable transcripts from the 5' or 3' flanking regions of these genes.

Animals↗

PubMed1996

RIGS (repeat-induced gene silencing) in Arabidopsis is transcriptional and alters chromatin configuration.

We have previously reported repeat-induced gene silencing (RIGS) in Arabidopsis, in which transgene expression may be silenced epigenetically when repeated sequences are present. Among an allelic series of lines comprising a primary transformant and various recombinant progeny carrying different numbers of drug resistance gene copies at the same locus, silencing was found to depend strictly on repeated sequences and to correlate with an absence of steady-state mRNA. We now report characterization, in nuclei isolated from the same transgenic lines, of gene expression by nuclear run-on assay and of chromatin structure by nuclease protection assay. We find that silencing is correlated with absence of run-on transcripts, indicating that expression is silenced at the level of transcription. We find further that silencing is also correlated with increased resistance to both DNase I and micrococcal nuclease, indicating that the silenced state reflects a change in chromatin configuration. We propose that silencing results when a locally paired region of homologous repeated nucleotide sequences is flanked by unpaired heterologous DNA, which leads chromatin to adopt a local configuration that is difficult to transcribe, and possibly akin to heterochromatin.

Arabidopsis↗

PubMed1996

The 5' nontranslated region of potato virus X RNA affects both genomic and subgenomic RNA synthesis.

A tobacco protoplast system was developed to analyze cis-acting sequences required for potato virus X (PVX) replication. Protoplasts inoculated with transcripts derived from a PVX cDNA clone or from clones containing mutations in their 5' nontranslated regions (NTRs) were assayed for RNA production by S1 nuclease protection assays. A time course of plus- and minus-strand-RNA accumulation indicated that both minus- and plus-strand PVX RNAs were detectable at 0.5 h postinoculation. Although minus-strand RNAs accumulated more rapidly than plus-strand RNAs, maximum levels of plus-strand RNAs were 40- to 80-fold higher. On the basis of these data, time points were chosen for determination of RNA levels in protoplasts inoculated with PVX clones containing deletions or an insertion in their 5' NTRs. Deletions of more than 12 nucleotides from the 5' end, internal deletions, and one insertion in the 5' NTR resulted in substantially decreased levels of plus-strand-RNA production. In contrast, all modified transcripts were functional for minus-strand-RNA synthesis, suggesting that elements in the 5' NTR were not essential for minus-strand-RNA synthesis. Further analysis of the 5' NTR deletion mutants indicated that all mutations that decreased genomic plus-strand-RNA synthesis also decreased synthesis of the two major subgenomic RNAs. These data indicate that cis-acting elements from different regions of the 5' NTR are required for plus-strand-RNA synthesis and that this process may be linked to synthesis of subgenomic RNAs.

Base Sequence↗

PubMed2000

Homocysteine stimulates nuclear factor kappaB activity and monocyte chemoattractant protein-1 expression in vascular smooth-muscle cells: a possible role for protein kinase C.

Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that stimulates the migration of monocytes into the intima of arterial walls. Although many factors that induce MCP-1 expression have been identified, the effect of homocysteine on the expression of MCP-1 in atherogenesis and the underlying mechanisms are not entirely clear. The objective of the present study was to investigate the role of homocysteine in MCP-1 expression in human aorta vascular smooth-muscle cells (VSMCs). After VSMCs were incubated with homocysteine for various time periods, a nuclease protection assay and ELISA were performed. Homocysteine (0.05-0.2 mM) significantly increased the expression of MCP-1 mRNA (up to 2. 7-fold) and protein (up to 3.3-fold) in these cells. The increase in MCP-1 expression was associated with the activation of protein kinase C (PKC) as well as nuclear factor kappaB (NF-kappaB). Further investigation demonstrated that the activation of NF-kappaB was the result of a PKC-mediated reduction in the expression of inhibitory protein (IkappaBalpha) mRNA and protein in homocysteine-treated cells. Oxidative stress might also be involved in the activation of NF-kappaB by homocysteine in VSMCs. In conclusion, the present study has clearly demonstrated that the activation of PKC as well as superoxide production followed by activation of NF-kappaB is responsible for homocysteine-induced MCP-1 expression in VSMCs. These results suggest that homocysteine-stimulated MCP-1 expression via NF-kappaB activation may play an important role in atherogenesis.

Blotting, Western↗

PubMed2001

Constitutive expression of functional GABA(B) receptors in mIL-tsA58 cells requires both GABA(B(1)) and GABA(B(2)) genes.

Studies of gamma-aminobutyric acid (GABA)(B) receptor function in heterologous cell systems have suggested that expression of two distinct seven transmembrane G-protein coupled receptor subunits is necessary for receptor activation and signal transduction. Some results suggest that both receptor proteins must be inserted into the plasma membrane to create heterodimers; however, it is possible that subunit monomers or homodimers are functional in cells which constitutively express GABA(B) receptors. A new pituitary intermediate lobe melanotrope cell clone (mIL tsA58) has been isolated which constitutively expresses GABA(B), D(2) and corticotrophin releasing factor receptors. Here, we report on characterization of the GABA(B) receptors. Solution hybridization-nuclease protection assays reveal the presence of GABA(B(1)) and GABA(B(2)) transcripts. Western blots show GABA(B(1a)) and one of two GABA(B(2)) proteins. Addition of the GABA(B) agonist baclofen to cultured mIL-tsA58 (mIL) cells inhibits high voltage activated Ca(2+) channels, as measured by agonist-induced inhibition of the K(+)-depolarization-stimulated increase in Ca(2+) influx. CGP55845, a GABA(B) antagonist, blocks the response to baclofen. Knockdown of either GABA(B(1)) or GABA(B(2)) subunits with selective antisense oligodeoxynucleotides reduced GABA(B) protein levels and completely abolished the GABA(B) receptor response in the mIL cells. Taken together, these results indicate that functionally active GABA(B) receptors in mIL cells require the constitutive expression of both GABA(B) genes. This is a physiologic validation of results from recombinant overexpression in naive cells and shows that the mIL cell line is a useful model for studying GABA(B) receptor expression, regulation and function.

Animals↗

PubMed1995

Regional quantification of dopamine transporter mRNA in rat brain using a ribonuclease protection assay.

We describe the regional distribution of dopamine transporter (DAT) mRNA in selected regions of rat brain using a highly sensitive and specific nuclease protection assay. This method determines the absolute quantity of mRNA expressed in the brain regions surveyed. DAT mRNA level varied widely between brain regions, and was detected only in cell body regions of the major dopaminergic pathways. Highest expression was seen in substantia nigra/ventral tegmentum (SN/VTA). Lower but detectable expression of a protected mRNA of the expected size was observed within hypothalamus. Expression could not be detected by this method in other brain regions studied. Our results indicate that this method is sufficiently sensitive to allow study of mRNA expression in individual animals.

Amino Acid Isomerases↗

PubMed1988

A role for divalent cations in specifying the start site for transcription from chromatin templates in vitro.

An assay that employs nucleoside 5'-O-(2-thiotriphosphates) was used to detect initiation of mouse mammary tumor virus (MMTV) RNA chains in preparations of isolated nuclei from cultured rat hepatoma cells containing stably integrated proviruses. RNA chains initiated with adenosine 5'-O-(2-thiotriphosphate), guanosine 5'-O-(2-thiotriphosphate), or uridine 5'-O-(2-thiotriphosphate) were separated from the remaining RNA by mercury-Sepharose column chromatography and analyzed for correctly initiated RNA chains with a T1 nuclease protection assay. Combined use of the thionucleotide transcription reaction with the T1 nuclease assay allowed precise localization of the transcription start sites. The majority of MMTV RNA chains were initiated with guanosine 5'-O-(2-thiotriphosphate) at a template site 133 nucleotides upstream from a PvuII site that coincides with the right end of the long terminal repeat. However, some RNA chains were also initiated with adenosine 5'-O-(2-thiotriphosphate) and uridine 5'-O-(2-thiotriphosphate) at template sites within three nucleotides of the primary guanosine start site. When Mn2+ was substituted for Mg2+ in the transcription reaction, MMTV RNA chains were initiated with approximately the same efficiency, but the start site was shifted to a position approximately 40 nucleotides downstream from the physiological start site; in the presence of Mn2+, MMTV RNA chains were initiated only with guanosine 5'-O-(2-thiotriphosphate). When the nuclei were exposed to both Mn2+ and Mg2+, transcription initiated at the manganese-dependent site. Mn2+ also caused the transcription start site for 45 S pre-rRNA to shift about 10 nucleotides upstream from the physiologically correct start site.

Animals↗

PubMed1990

A solution hybridization/RNase protection assay with riboprobes to determine absolute levels of apoB, A-I, and E mRNA in human hepatoma cell lines.

A solution hybridization/RNase protection assay with riboprobes was developed to quantitate apolipoprotein mRNA concentrations. Previously, radiolabeled DNA probes have been used in solution hybridization/S1 nuclease protection assays for this purpose. The new assay requires less time for probe preparation and hybridization compared to previous assays. In addition, the vector used for riboprobe preparation can also be used to conveniently produce cRNA required to generate the standard curve to quantitate absolute apolipoprotein mRNA levels. The solution hybridization RNase protection assay was used to quantitate apoB, A-I, and E mRNA levels in four human hepatoma cell lines, HepG2, Hep3B, WRL-68, SK-Hep2. HepG2 and Hep3B, but not WRL-68 and SK-Hep2 cells had concentrations of all three apolipoprotein mRNAs comparable to liver in vivo. These data suggest that HepG2 and Hep3B are suitable models to study liver specific apolipoprotein gene expression.

Apolipoprotein A-I↗