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Results for “Nuclease Protection Assays”

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PubMed1991

Differential regulation of insect globin and actin mRNAs during larval development in Chironomus thummi.

S1 nuclease protection assays were used to measure changes in the steady-state levels of six different globin (Gb) mRNAs in the midge, Chironomus thummi thummi (C. thummi, Diptera) during larval development. Two distinct patterns of change were observed. GbI, IV, VIIB-4 and VIIB-5 transcripts were present in 3rd instar larvae, rose from low levels immediately post-moult to peak levels by day 2-3 of the 4th instar, and then declined, reaching near-basal levels by day 7-8. In contrast, transcripts of GbIII (known from previous studies to be 4th instar-specific) and VI, which were undetectable in the 3rd instar, rose to high levels by day 2 of the 4th instar, but remained elevated thereafter. Our data further showed that closely linked Gb genes were not necessarily expressed in a coordinate manner. Unlike the Gb mRNAs, actin (Act) mRNA levels (measured by slot-blot hybridization to a heterologous probe) increased progressively as a proportion of total RNA during 4th instar development. Therefore, the regulation of C. thummi Gb transcript levels is specific, differing from that of Act and among the Gb mRNAs themselves. Elevated 20-hydroxyecdysone (HE) titer at the 3rd-4th instar moult correlates with the low steady-state levels of Gb mRNAs immediately post-moult. However, other aspects of Gb mRNA profiles cannot be explained on the basis of a direct repressive effect by HE on Gb gene transcription.

Actins↗

PubMed1992

Structure of the rat gene encoding the mitochondrial benzodiazepine receptor.

The gene encoding the rat mitochondrial benzodiazepine receptor (MBR) was cloned and characterized. Hybridization of a previously cloned cDNA for MBR to genomic Southern blots indicated that the gene was probably present at one copy per haploid genome. Rapid amplification of cDNA ends with rat adrenal RNA was used to obtain 47 nt of additional sequence upstream from our previously cloned MBR cDNA proving to be a crucial step in cloning the first exon of this gene. The MBR gene is comprised of four exons spanning approx. 10 kb. The first intron, contained within a 8-kb stretch of this gene, is located within the 5'-untranslated sequence, whereas the remaining two introns are much shorter (641 and 854 bp) and interrupt the coding sequence. The third intron contains sequences homologous to rodent B1 repetitive elements and a novel sequence closely resembling part of a repetitive element belonging to the Alu family in humans. The transcription start point was mapped by S1 nuclease protection assays suggesting that the first exon is just 56 bp in length. The sequence upstream from this region contains three GC boxes but lacks other known consensus recognition sites for sequence-specific transcription factors.

Amino Acid Sequence↗

PubMed1994

In vitro transcription of the TATAA-less mouse thymidylate synthase promoter: multiple transcription start points and evidence for bidirectionality.

The mouse thymidylate synthase (TS) promoter (pTS) lacks a TATAA box and an initiator element, and has multiple transcription start points (tsp) located across a 90-bp region. We have developed an in vitro transcription system for pTS using circular templates and nuclear extracts from HeLa cells or mouse 3T6 fibroblasts. The amount of RNA synthesized and the locations of the tsp were determined by S1 nuclease protection assays. The transcription system reproduced the complex pattern of in vivo tsp, except that the downstream tsp were used preferentially. The reaction temperature, concentrations of DNA template and MgCl2, and incubation time were optimized. The pTS core region contains binding sites for the Sp1 and Ets transcription factors. Inactivation of the Sp1-binding element led to a twofold reduction in transcription and a preferential use of upstream tsp. Inactivation of the Ets-binding element, which reduced promoter activity tenfold in vivo, had only a minor effect in vitro. Addition of a strong initiator element introduced a new tsp, but did not eliminate the complex tsp pattern. To determine if pTS had bidirectional promoter activity, the promoter was inverted and analyzed for transcriptional activity. The inverted promoter was found to initiate transcription at multiple tsp and had approximately the same strength as the normal pTS.

Animals↗

PubMed1995

A unique intron-containing hsp70 gene induced by heat shock and during sporulation in the aquatic fungus Blastocladiella emersonii.

We have isolated and characterized cDNA and genomic DNA clones encoding the 70-kDa heat-shock protein (Hsp70) from the aquatic fungus Blastocladiella emersonii (Be). Nucleotide (nt) sequence analysis predicts an acidic protein containing 650 amino acids, with a calculated molecular mass of 70.8 kDa. The Be hsp70 gene is induced by heat shock (HS), as well as during sporulation of the fungus, and its coding region is interrupted by a single intron. All the evidence seems to indicate that this is the only hsp70 in Be. S1 nuclease protection assays revealed that splicing of the hsp70 intron is highly thermoresistant; at the lethal temperature of 42 degrees C, only 30% of the hsp70 mRNAs have not been processed. A single transcription start point (tsp), localized about 30 nt downstream from a putative TATA box, was determined both during HS and at normal temperatures. The promoter region presented several NGAAN repeats (where N is any nucleotide) characteristic of HS elements, as well as putative binding sites for ATF, Sp1 and two metal-responsive elements.

Amino Acid Sequence↗

PubMed1989

Level of expression and chromosome mapping of the mouse cholecystokinin gene: implications for murine models of genetic obesity.

Cholecystokinin (CCK) is a neuropeptide which is present in brain and intestine and which stimulates gall bladder contraction and pancreatic secretion. Additional studies have demonstrated an appetite-suppressing effect of CCK in vivo. These data have aroused speculation that the physiology of this hormone could be relevant in the pathogenesis of the mouse obesity mutations ob on chromosome 6 and db on chromosome 4. In order to determine whether abnormalities of this hormone could be the primary defect in these obesity mutations, we have used three separate approaches to map the mouse Cck gene to distal chromosome 9, where it is part of a syntenic group between mouse chromosome 9 and human chromosome 3. These data therefore exclude cholecystokinin as the etiologic factor in the pathogenesis of any of the known mouse obesity syndromes. In order to exclude the possibility that there are differences in mutant animals in the level of CCK RNA, we have used an S1 nuclease protection assay as well as a novel radioimmunoassay that detects the CCK precursor, to show that there are no gross differences in CCK mRNA or protein precursor levels between ob/ob and wild-type animals.

Alleles↗

PubMed1994

Regulation of intracellular cholesterol metabolism is defective in lymphoblasts from Niemann-Pick type C and type D patients.

Regulation of intracellular cholesterol metabolism has been studied in Epstein-Barr virus-transformed lymphoblasts from patients with Niemann-Pick type C (NPC) and the Nova Scotia type D (NPD) disease. Addition of LDL to normal lymphoblasts cultured in lipoprotein-deficient medium increased cholesterol esterification 10-fold (to a maximum of 1.0 nmol/h/mg protein at 15 h), while little stimulation was seen in NPC cells. The response by NPD lymphoblasts was intermediate, reaching approximately half of normal values by 14-24 h. Lymphoblasts from both NPC and NPD obligate heterozygotes exhibited 50% of normal LDL-stimulated cholesterol esterification at 6 h, when activity was < 10% of normal values in patient cells. Fluorescence staining with filipin indicated excessive intracellular accumulation of LDL-derived cholesterol in both NPC and NPD lymphoblasts. Downregulation of LDL receptor mRNA levels by LDL, measured by S1 nuclease protection assay, was also impaired in NP lymphoblasts and fibroblasts (NPC > NPD), although a similar rate of receptor protein down-regulation by LDL (t1/2 = 10-15 h) was observed in normal and NP lymphoblasts. In contrast, LDL down-regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA did not appear to be affected in NP cells: LDL produced a 3-fold (lymphoblasts) or > 10-fold (fibroblasts) decrease by 12 h in both normal and affected cells. Thus, NPC and NPD lymphoblasts exhibit distinct defects in cholesterol esterification and storage, similar to those observed in mutant fibroblasts. Other regulatory responses are also impaired in NPC lymphoblasts but appear to be less affected in NPD cells. Lymphoblasts should provide a valuable immortalized cell line model for study of defective regulation of cholesterol esterification and transport in Niemann-Pick type II disease, and may also be suitable for diagnosis and carrier detection.

Cholesterol↗

PubMed2005

Cloning of the growth hormone cDNA of alligator gar Atractosteus spatula and its expression through larval development.

The alligator gar (Atractosteus spatula) is the largest freshwater fish inhabiting rivers draining into the Gulf of Mexico. This primitive fish shows a fast growth rate since its early larval stages. This is attributed to the action of growth hormone (GH), an anterior pituitary gland hormone responsible for linear growth in vertebrates that can also be expressed in extrapituitary adult tissues and in fish embryos. The present research was aimed at obtaining the GH coding sequence of the alligator gar and studying its expression through larval development. A cDNA was obtained by RT-PCR, cloned and sequenced. The alligator gar GH cDNA sequence shares 98% nucleotide similarity with that reported for Lepisosteus osseus, indicating a very slow evolution of the GH within the primitive fish, in contrast with the burst of changes observed in euteleosts. Using RT-PCR and RNA nuclease protection assays, GH transcripts were detected at very high levels in eggs, embryos and in several larval stages. These data suggest that the GH may play an important role during embryogenesis in fish. The better understanding of alligator gar larval physiology will facilitate the culture of larvae and juvenile gar and consequently may allow the restoration of their natural populations.

Alligators and Crocodiles↗

PubMed2005

Bacillus subtilis bacteriophage SPP1 G40P helicase lacking the n-terminal domain unwinds DNA bidirectionally.

Bacillus subtilis bacteriophage SPP1 G40P hexameric replicative DNA helicase unidirectionally translocates with a 5'-->3' polarity while separating the DNA strands. A G40P mutant derivative lacking the N-terminal domain (containing amino acid residues 110-442 from G40P, G40PDeltaN109) was purified and characterized. G40PDeltaN109 showed an ATPase activity that was dependent on the presence of single-stranded (ss) DNA. Unlike G40P, G40PDeltaN109 was shown to bind with similar affinity both ssDNA arms of forked structures by nuclease protection assays. In a pH-dependent manner, G40PDeltaN109 unwound a branched double-arm substrate preferentially with a 3'-->5' polarity. Our results show that the linker region and the C-terminal domain of G40P are sufficient to render an enzyme capable of encircling the ssDNA tails of the forked DNA and to unwind DNA with both 5'-->3' and 3'-->5' polarity. The presence of the N-terminal domain, which does not play an essential role in helicase action, might be required indirectly for strand discrimination and polarity of translocation.

Adenosine Triphosphate↗

PubMed2004

Neurokinin-1 receptor gene expression in the mouse dorsal horn increases with neuropathic pain.

UNLABELLED: Peripheral nerve injury is associated with hyperesthesia and increased neurokinin-1 receptor (NK-1) expression in the dorsal horn of the spinal cord. To test the hypothesis that NK-1 gene expression underlies these responses, we used solution hybridization-nuclease protection assays to quantify NK-1 mRNA levels in dorsal quadrants of the mouse lumbar dorsal horn. Partial sciatic nerve ligation was associated with mechanical allodynia, thermal hyperalgesia, and an increase in NK-1 mRNA on the ipsilateral, but not contralateral, side. Regression analysis showed that NK-1 mRNA was significantly correlated with thermal paw withdrawal latency but not mechanical threshold. Our results support the idea that substance P is an important mediator of thermal hypersensitivity in the setting of nerve injury and suggest that increased NK-1 receptor transcription precedes increased NK-1 receptor density, ultimately leading to behavioral hypersensitivity to peripheral thermal stimulation. PERSPECTIVE: The therapeutic efficacy of NK-1 receptor antagonists is unclear. The current data suggest that peripheral nerve injury increases the expression of substance P (NK-1) receptors in the spinal cord dorsal horn; this is correlated with heat hypersensitivity. The analgesic effects of NK-1 antagonists might become apparent if tested against heat-evoked pain in nerve injury patients.

Animals↗

PubMed2005

Role of lymph-borne cells in the early stages of scrapie agent dissemination from the skin.

Scrapie is a natural transmissible spongiform encephalopathy (TSE) of sheep, infecting the animal via the gastrointestinal tract or the skin. This project tested the hypotheses that lymph-borne cells (especially dendritic cells) are crucial for the systemic dissemination of the infectious agent from the site of infection in the skin, that PrP genotype affects PrPSC association with dendritic cells and that PrPSC carriage by cells affects their expression of cytokines. Skin, of scrapie-susceptible VRQ/ARR and scrapie-resistant ARR/ARR PrP genotypes, was scarified with FITC-labelled PrPSC. Pseudoafferent lymphatic cannulation was then used to monitor the presence of FITC-PrPSC over time in different lymph cell populations and plasma in the draining afferent lymphatics. The major observation was that PrPSC did not associate significantly with any lymphocyte or dendritic cell population in the 5 days following PrPSC scarification. The only cells seen to associate with PrPSC were neutrophils. Furthermore, despite the quantity of PrPSC used for scarification being equivalent to a standard infectious dose (the VRQ/ARR sheep dying at approximately 260 days) the only PrP found in afferent lymph during the 0-5-day period was proteinase K sensitive (i.e. soluble PrPC). No differences were observed between the PrP genotypes. Analysis of the effects of PrPSC scarification of cellular cytokine mRNA expression (by a nuclease protection assay) showed raised levels of IL-1beta and IL-8 in the susceptible VRQ/ARR group and raised levels of IFNgamma in the resistant ARR/ARR animals.

Animals↗

PubMed1992

Localization of in vivo ribosome pause sites.

A protocol for the localization of the 5' boundaries of in vivo ribosomal pausing sites has been developed. These mapping experiments combine two basic techniques. The first is the isolation of polysomal transcripts via centrifugation of tissue extracts through a sucrose cushion in the presence of translational elongation inhibitors. The second technique involves a micrococcal nuclease protection assay first developed by Wolin and Walter for in vitro-bound ribosomes (EMBO J. 7, 3559-3569, 1988). Using this method, the 5' boundaries of in vivo ribosomal pause sites were localized on spinach chloroplast mRNAs derived from the atpA gene. This method is easily adaptable to the identification of in vivo ribosomal pause sites from any organism. It could also be adapted to the localization of in vivo binding sites for other nucleic acid binding proteins.

Base Sequence↗

PubMed2001

Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures.

Typical methods used for encapsulating antisense oligodeoxynucleotides (ODN) and plasmid DNA in lipid vesicles result in very low encapsulation efficiencies or employ cationic lipids that exhibit unfavorable pharmacokinetic and toxicity characteristics when administered intravenously. In this study, we describe and characterize a novel formulation process that utilizes an ionizable aminolipid (1,2-dioleoyl-3-dimethylammonium propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities (0.15--0.25 g ODN/g lipid) of polyanionic ODN in lipid vesicles. This process requires the presence of up to 40% ethanol (v/v) and initial formulation at acidic pH values where the DODAP is positively charged. In addition, the presence of a poly(ethylene glycol)-lipid was required during the formulation process to prevent aggregation. The 'stabilized antisense-lipid particles' (SALP) formed are stable on adjustment of the external pH to neutral pH values and the formulation process allows encapsulation efficiencies of up to 70%. ODN encapsulation was confirmed by nuclease protection assays and (31)P NMR measurements. Cryo-electron microscopy indicated that the final particles consisted of a mixed population of unilamellar and small multilamellar vesicles (80--140 nm diameter), the relative proportion of which was dependent on the initial ODN to lipid ratio. Finally, SALP exhibited significantly enhanced circulation lifetimes in mice relative to free antisense ODN, cationic lipid/ODN complexes and SALP prepared with quaternary aminolipids. Given the small particle sizes and improved encapsulation efficiency, ODN to lipid ratios, and circulation times of this formulation compared to others, we believe SALP represent a viable candidate for systemic applications involving nucleic acid therapeutics.

Animals↗

PubMed2003

HrcA is a negative regulator of the dnaK and groESL operons of Streptococcus pyogenes.

The genome of Streptococcus pyogenes, an important human pathogen, encodes homologs of the principal bacterial heat shock proteins DnaK and GroES, -EL, as well as HrcA, a negative regulator of dnaK and groESL expression in other Gram-positive bacteria. Using nuclease protection assays to measure dnaK/groESL mRNA abundance and a "non-polar" insertion to disrupt hrcA, we demonstrate that heat shock triggers a 4- to 8-fold increase in dnaK and groESL-specific mRNAs within 5 min of the temperature shift and that HrcA is a negative regulator of S. pyogenes dnaK/groESL mRNA abundance in unstressed S. pyogenes. Although the loss of HrcA elevated dnaK and groESL mRNA levels under non-heat shock conditions, the relative abundance of these RNAs increased further in heat shocked S. pyogenes, suggesting an additional element contributing to their synthesis or stability.

Bacterial Proteins↗

PubMed1991

Structural differences in 5'-flanking regions of rat cytochrome P-450aldo and P-450(11) beta genes.

Two rat genomic clones, one for cytochrome P-450aldo and the other for P-450(11) beta, were isolated and characterized. The two genes, encoding structurally homologous proteins, were closely similar in their intron-exon organizations. Their 5'-flanking regions, however, contained only a few homologous regions. A putative cyclic AMP responsive element, TGACGTGA, was found in the P-450aldo gene, but this sequence was altered at two positions in the P-450(11) beta gene. S1 nuclease protection assay revealed a single transcription initiation site for the P-450aldo gene, while multiple sites were found for the P-450(11) beta gene. These results suggest that transcriptional regulation of the rat P-450aldo and P-450(11) beta genes is due to differences in the sequences of their 5'-flanking regions.

Animals↗

PubMed2001

Inhibition by ginkgolides and bilobalide of the production of nitric oxide in macrophages (THP-1) but not in endothelial cells (HUVEC).

Nitric oxide (NO) is a principal mediator in many physiological and pathological processes. NO produced by constitutive nitric oxide synthase in endothelial cells (eNOS) acts as a vasodilator, whereas excess NO production due to elevated expression of inducible nitric oxide synthase (iNOS) may produce cytotoxic effects to cells in the vascular wall. We demonstrated in our previous work that the extract of Ginkgo biloba leaves (EGb) inhibits iNOS-mediated NO production. The objective of the present study was to investigate the effects of several active EGb components on iNOS-mediated NO production in macrophages derived from a human monocytic cell line (THP-1), as well as on eNOS-mediated NO production in human umbilical vein endothelial cells (HUVEC). Ginkgolide A, ginkgolide B, or bilobalide (0.25 to 1.0 microg/mL) caused a 30-65% reduction in the levels of NO metabolites released by THP-1 macrophages after 4 hr of incubation, with a corresponding decrease in iNOS activity. Western immunoblotting analysis coupled with a nuclease protection assay and reverse transcription-polymerase chain reaction revealed a concomitant reduction in the levels of iNOS protein mass and mRNA in ginkgolide A-, ginkgolide B-, or bilobalide-treated macrophages. On the other hand, these compounds did not affect eNOS-mediated NO production or the expression of eNOS protein and mRNA in HUVEC. Taken together, these results suggest that ginkgolide A, ginkgolide B, and bilobalide may contribute to the selective inhibitory effect of EGb on iNOS expression without affecting eNOS-mediated NO production.

Cells, Cultured↗

PubMed2001

Spatial learning induces neurotrophin receptor and synapsin I in the hippocampus.

We report that rats learning a spatial memory task in the Morris water maze show elevated expression of the signal transduction receptor for BDNF and the synaptic associated protein synapsin I in the hippocampus. Nuclease protection assays showed maximal levels of TrkB and synapsin I mRNAs in the hippocampus by the time that asymptotic learning performance had been reached (Day 6). Increases in synapsin I mRNA were matched by changes in synapsin I protein as revealed by western blot analysis. Synapsin I is a downstream effector for the BDNF tyrosine kinase cascade pathway which has important roles in synaptic remodeling and function. Therefore, parallel changes in TrkB and synapsin I mRNAs suggest a role of the BDNF system in synaptic function or adaptation. Levels of TrkB mRNA in the hippocampus were attenuated after learning acquisition (Day 20), but synapsin I mRNA was still elevated, suggesting that the BDNF system may participate in events secondary to learning, such as strengthening of neural circuits. TrkB and synapsin I mRNAs showed an increasing trend in the cerebellum of learning rats and no changes were observed in the caudal cerebral cortex. The selectivity of the changes in trkB and synapsin I, affecting the hippocampus, is in agreement with the role of this structure in processing of spatial information. Behavioral regulation of neurotrophins may provide a molecular basis for the enhanced cognitive function associated with active lifestyles, and guide development of strategies to promote neural healing after CNS injury or disease.

Animals↗

PubMed2002

Effects of adrenalectomy on AGRP, POMC, NPY and CART gene expression in the basal hypothalamus of fed and fasted rats.

Glucocorticoids regulate body energy balance through both peripheral and central mechanisms. In order to understand the central mechanisms that mediate these effects of glucocorticoids we studied the effects of adrenalectomy (ADX) and food deprivation on the expression of four neuropeptide genes (measured by S1 nuclease protection assay) in the medial basal hypothalamus (MBH), which are known to regulate energy balance: pro-opiomelanocortin (POMC), agouti-related peptide (AGRP), neuropeptide Y (NPY), and cocaine and amphetamine regulated transcript (CART). Adult male rats were ADX or sham operated (SHAM), and studied 1-2 weeks later. In the first study effects of ADX and corticosterone replacement on POMC and AGRP expression were determined. ADX decreased POMC and AGRP gene expression in the MBH by 27 and 38%, respectively, compared to SHAM rats. Corticosterone treatment increased the expression of POMC by 87% and AGRP by 45% in ADX rats. The second study was designed to determine if glucocorticoids are necessary for the fasting induced changes in POMC, AGRP, NPY and CART in the MBH. ADX caused a 20-30% decrease in the expression of all four neuropeptide genes in the MBH. As expected, fasting suppressed POMC and CART expression and increased AGRP and NPY expression. The fasting-induced increases in AGRP and NPY persisted after ADX but no further significant decreases in POMC or CART were noted after fasting in ADX rats. Plasma leptin and insulin declined significantly after ADX and increased with corticosterone replacement; both leptin and insulin declined further in fasted, ADX animals. In conclusion, ADX decreases both anorexigenic, POMC and CART, and orexigenic, AGRP and NPY, neuropeptide gene expression in the MBH. AGRP and NPY decrease after ADX despite the fall in plasma leptin and insulin concentrations which in other situations would increase these neuropeptides. Furthermore, glucocorticoids are not required for fasting-induced upregulation of AGRP and NPY expression.

Adrenal Cortex↗

PubMed2003

Formalin- or adjuvant-induced peripheral inflammation increases neurokinin-1 receptor gene expression in the mouse.

Substance P (SP) has been widely studied as a mediator of nociception. The release of SP from primary afferent neurons is increased during nociception, and SP activates neurokinin-1 (NK-1) receptors in the spinal cord and periphery. Nociception-evoked alterations in NK-1 receptor gene expression have been studied in rat models of persistent pain but have not been characterized in any murine models of peripheral inflammation. This study assessed behavioral responses and NK-1 receptor mRNA gene expression in mice receiving formalin or Freund's complete adjuvant (CFA) as an inflammatory stimulus. Mechanical withdrawal thresholds were measured before injection of formalin or CFA and hind paw licking/biting timed during the late-phase of the formalin response. Two and 24 hours after formalin or CFA injection, mechanical withdrawal thresholds were measured and the mice euthanized. Solution hybridization-nuclease protection assays were used to quantify NK-1 receptor mRNA levels. Results demonstrated that inflamed hind paws were edematous, and the withdrawal thresholds of the inflamed hind paws were significantly lower after formalin or CFA injection. Neurokinin-1 receptor mRNA levels in the ipsilateral dorsal spinal cords of mice were higher at 24 h after formalin injection or 4 days after CFA injection. These results confirm that mice are hyperalgesic at late time points after formalin or adjuvant injection when NK-1 receptor gene expression is elevated in the dorsal spinal cord. This supports the hypothesis that increased NK-1 receptor gene expression contributes to the development and maintenance of a hyperalgesic state.

Animals↗