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Results for “Nuclease Protection Assays”

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At least 145 records · Page 8Linked to original sources

PubMed1994

Expression and alternative splicing of the neural cell adhesion molecule NCAM in human granulosa cells during luteinization.

Freshly aspirated human granulosa cells from pre-ovulatory follicles and granulosa cells luteinized in culture possess the neural cell adhesion molecule (NCAM) of approximate molecular mass of 140,000 and NCAM mRNA as confirmed by S1-nuclease protection assays and RT-PCR. Moreover, in the process of luteinization the NCAM isoform pattern is modified. Isoforms containing an insert of 10 amino acids (termed VASE) in the extracellular domain of NCAM were supplemented by alternatively spliced isoforms without this insert. NCAM immunoreactivity, at light and electron microscope levels, was associated with the cell membrane of most granulosa cells which formed clusters. During time in culture an increasing subpopulation of granulosa cells, devoid of NCAM immunoreactivity, spread out and formed monolayers. This differential expression and the alternative splicing of NCAM during luteinization of granulosa cells raise the possibility that NCAM could be involved in folliculogenesis and the formation of the corpus luteum in the human.

Alternative Splicing↗

PubMed1989

Expression of substance P/neurokinin A-encoding preprotachykinin messenger ribonucleic acids in the rat enteric nervous system.

The cellular localization of substance P/neurokinin A-encoding preprotachykinin mRNAs in the rat enteric nervous system was studied by means of in situ hybridization histochemistry using 35S- or 3H-labeled single-stranded ribonucleic acid (RNA) probes which recognize all three preprotachykinin mRNA species, alpha, beta, and gamma. Substance P/neurokinin A-encoding mRNAs are expressed in neurons within the myenteric plexus of the esophagus and stomach, being more numerous in the latter, and in ganglion cells distributed to both the myenteric and submucosal plexuses of the intestine. Specificity of the hybridization was demonstrated by the lack of specific signal above background in sections incubated with a sense RNA probe or pretreated with ribonuclease A before hybridization. Ribonucleic acid blot hybridization analysis of RNA extracts from both the muscle layer-myenteric plexus and submucosal layer preparations of the duodenum demonstrated a single band of hybridization at 1.3 kb. Solution hybridization-nuclease protection assays showed multiple preprotachykinin-encoding transcripts in these RNA extracts, with an abundance level of gamma-mRNA greater than beta-mRNA much greater than alpha-mRNA, which is similar to that observed in the rat brain. Our results indicate that the preprotachykinin gene encoding the tachykinin peptides, substance P and neurokinin A, is transcribed in a population of enteric neurons that have a regional distribution comparable to the previously described tachykinin-like immunoreactive neurons, suggesting that specific mRNAs and the posttranslationally processed peptides are localized in the same structures.

Animals↗

PubMed1986

Distinct sequence elements involved in the glucocorticoid regulation of the mouse mammary tumor virus promoter identified by linker scanning mutagenesis.

In the proviral DNA of mouse mammary tumor virus (MMTV), sequences up to approximately equal to 200 base-pairs from the RNA start site are required for stimulation of transcription by glucocorticoid hormones in cultured cells. A total of 26 mutant plasmids with clustered point mutations or small deletions in the hormone control region of the MMTV long terminal repeat were constructed, linked to the coding portion of the Herpes simplex virus thymidine kinase gene, and introduced by transfection into LTK- cells. Transcription from mutant DNA in the presence or absence of hormone was quantified by S1 nuclease protection assays. Our analysis revealed the presence of at least three control elements that affect the extent of transcription stimulation by glucocorticoid hormones: (1) a distal element, between -181 and -172 base-pairs from the RNA initiation site. Linker scanning mutants in this segment have a reduction of up to 20-fold in the hormone response with respect to wild type. (2) An element around position -120, defined by a mutation of 4 base-pairs between -121 and -117, which causes a fivefold reduction. (3) An element from approximately equal to -78 to -70, defined by a mutant with also a roughly fivefold lower stimulation. The first two are included in areas that have been shown by others to interact in vitro with hormone-receptor complexes; the last one overlaps the in vitro binding site of a nuclear protein factor. A mutant lacking all three elements (-193 to -70) is completely non-inducible by glucocorticoids. Together with earlier results obtained with 5' deletion mutants, the data show that the largest contribution to the stimulatory response is made by the distal element, which however does require the presence of both more-proximal ones for the response to be maximal. In the absence of the distal one, the two proximal elements together produce a residual stimulation in the order of 5 to 10% of wild type, while the -70 element alone is ineffective. In addition, we show that a functional TATA homology is required for maximum stimulation. It appears that transcriptional regulation of MMTV by glucocorticoid hormones is achieved by the concerted action of multiple sequence modules, not all of which correspond to receptor binding sites in vitro.

Animals↗

PubMed1988

Interactions of Escherichia coli transcription termination factor rho with RNA. II. Electron microscopy and nuclease protection experiments.

Structural aspects of the interaction between Escherichia coli transcription termination factor rho and RNA have been investigated, using nuclease protection assays and electron microscopy. A synthetic RNA, poly(rC), has been used as a substrate for these studies, since it binds tightly to rho and acts as a strong activator of the ATPase activity of rho. Digestion of oligo(rC)-rho complexes with ribonuclease A yields oligo(rC) fragments with a maximum length of 70 to 80 nucleotide residues. Electron micrographs demonstrate that rho binds to poly(rC) as a toroid-shaped oligomer with an outside diameter of approximately 120 A. Taken together with data from the accompanying paper, which shows that the RNA binding site size per rho monomer is 13(+/- 1) nucleotide residues, we infer that rho binds RNA as a hexamer with an oligomeric site size of 72 to 84 residues. Further analysis of the electron micrographs has revealed that the polynucleotide chain is wrapped around, or condensed within, the protein oligomer. rho hexamers bind to poly(rC) with moderate co-operativity (omega = 380 +/- 60), displaying no significant preference for binding to chain ends versus internal sites on the polynucleotide chain. These findings and those of the companion paper are discussed in terms of various models for the structure of the rho-RNA complex in transcription termination.

Allosteric Site↗

PubMed1992

Cerebral cortical amyloid protein precursor mRNA expression is similar in Alzheimer's disease and other neurodegenerative diseases.

The expression of 3 beta-amyloid protein precursor (APP) mRNAs (695, 751, and 770) in the cerebral cortex in Alzheimer's disease and other neurodegenerative diseases was analyzed by the S1 nuclease protection assay. We found no significant Alzheimer's disease-specific alteration of APP mRNA expression when compared to the other neurological diseases as controls. Since the expression of this mRNA was not correlated with amyloid deposition, it is possible that gliosis/neuronal loss may secondarily alter APP mRNA expression. However, the current study revealed no significant correlation between them.

Adult↗

PubMed1989

Cell type specific expression and regulation of murine interferon alpha and beta genes.

The differential and cell type specific expression of various murine IFN alpha genes and IFN beta was examined by S1 nuclease protection assays in M-CSF cultured C57BL/6 mouse bone marrow macrophages and L929 fibroblasts. In Newcastle disease virus (NDV) induced macrophages, IFN beta, alpha 2, alpha 4, and alpha 1 mRNAs were the predominant species, whereas IFN alpha 6 and alpha 9 transcripts accounted for only 5% of total IFN alpha mRNAs. In L929 cells, only IFN beta, alpha 2, and alpha 4 genes were expressed efficiently following NDV induction and IFN alpha 9 mRNA was always below detectable level. Induction of macrophages with the synthetic inducer 10-carboxymethyl-9-acridanone resulted in small amounts of IFN alpha 2, alpha 4, and alpha 6 mRNAs and the IFN beta mRNA level was about 100-fold higher. Macrophages and L929 cells especially differed in the kinetics of IFN gene induction in that macrophages showed a much earlier transient expression of all IFN mRNA species. Additionally, IFN transcripts were degraded much faster in macrophage cultures than in L929cells. The IFN response of macrophages is thus characterized by a highly efficient control, providing a rapid onset and a rapid decline of IFN production, which limits release of IFN to a short time interval.

Animals↗

PubMed1991

Quantitative detection of spliced E6-E7 transcripts of human papillomavirus type 16 in cervical premalignant lesions.

The splicing patterns of E6-E7 transcripts of human papillomavirus type 16 (HPV16) in cervical premalignant lesions were quantitatively analyzed by S1 nuclease protection assay. The major E6-E7 transcripts in HPV16-containing cervical lesions (four cervical intraepithelial neoplasias and one invasive carcinoma) were from spliced E6*I/E7 mRNA. The unspliced E6/E7 mRNA, which can encode the full-length zinc finger protein E6, is expressed as 8 to 15% of E6-E7 transcripts. The spliced E6*II/E7 mRNAs were expressed as 14 to 24% of E6-E7 transcripts in most tissues. However, in HPV 16-containing cell lines, the expression levels of spliced and unspliced E6-E7 transcripts were variable.

Blotting, Southern↗

PubMed1991

Expression of measles virus RNA in peripheral blood mononuclear cells of patients with measles, SSPE, and autoimmune diseases.

In order to characterize measles virus (MV) infection in peripheral blood mononuclear cells (PBMCs), RNA was isolated from PBMCs after PHA-stimulation for 72 hr of 9 patients with acute measles, 16 patients with subacute sclerosing panencephalitis (SSPE), 13 patients with various autoimmune diseases, and 16 healthy control donors. The RNA obtained was screened for the presence of MV N (nucleocapsid) gene specific transcripts of either positive or negative orientation in a S1 nuclease protection assay. The sensitivity of this assay allowed us to detect one infected cell in 20,000 PBMCs or 0.1 to 0.05 copies of MV-specific RNA per cell. Using single-stranded DNA or RNA probes expression of MV genomic RNA of negative polarity could be detected in only one case of acute measles and one healthy control donor. Conversely, N-specific transcripts of positive polarity, indicating active transcription, could only be detected in patients with acute measles. In addition, in infected PBMCs and in a persistently MV-infected B cell line positive stranded N-specific transcripts containing leader usually present at very low frequency have been found in relatively increased amounts in comparison with transcripts lacking leader. Whereas the ratio of these RNA species during lytic infection with MV in Vero cells is about 1:50, the ratio found here ranges from 1:3 to 1:10. This altered ratio indicates a specific regulation of MV specific transcription in cells of lymphoid origin that has not been found in any other cell system analyzed.

Animals↗

PubMed1988

Antisense RNA inhibits expression of membrane skeleton protein 4.1 during embryonic development of Xenopus.

Plasmids expressing partial-length sense or antisense protein 4.1 RNA were microinjected into fertilized Xenopus eggs. Nuclease protection assays reveal that antisense protein 4.1 RNA lead to the specific loss of endogenous protein 4.1 transcripts after midblastula transition, with no effect on the levels of three unrelated transcripts. As a control, we show that this dramatic loss of endogenous protein 4.1 transcripts is blocked when fertilized eggs receive a second injection of plasmids that express partial-length sense protein 4.1 RNA. Immunocytochemistry of tadpole embryos with antibodies monospecific for protein 4.1 demonstrates that the antisense protein 4.1 RNA blocks the normal expression of protein 4.1 in embryos and interferes with the normal interdigitation of the photoreceptor outer segments with the pigment epithelium layer in the retina. These data suggest that reduced expression of a single membrane skeleton protein is sufficient to perturb normal cellular interactions of the retina.

Amino Acid Sequence↗

PubMed1990

NCAM gene expression during the development of cerebellum and dentate gyrus in the mouse.

The neural cell adhesion molecule (NCAM) is thought to be involved in several important events during CNS vertebrate development. This study provides additional information concerning the biochemical determination and anatomical localization of NCAM transcripts. Using S1 nuclease protection assays (S1-NPAs), NCAM transcripts in brain appear highest at birth, with NCAM messenger levels reduced some 20-fold by adulthood. By use of in situ hybridization, NCAM mRNA is demonstrated to be developmentally regulated in the cerebellum and hippocampus. The in situ hybridization findings, in addition to providing results to compare with past studies of NCAM immunolocalization, reveal that NCAM expression in dentate gyrus granule cells and cerebellar Purkinje cells is correlated with the final stages of axonal growth, e.g., synaptic stabilization. In situ hybridization demonstrates a developmental outside-to-inside gradient of NCAM transcripts in the dentate gyrus. Neurological mutant mice, reeler and stagger, provide evidence that NCAM expression is normal in the brain regions investigated, and does not correlate with the developmental perturbations present in these strains.

Animals↗

PubMed1992

The identification and characterization of a break within the large subunit ribosomal RNA of Trichinella spiralis: comparison of gap sequences within the genus.

A break was identified in the large subunit ribosomal RNA of Trichinella spiralis that results in its dissociation into 2 smaller fragments of approximately equal length. The approximate location of the break within the encoding gene was mapped from subcloned rDNA fragments by S1 protection experiments. The boundaries of the break were determined by cDNA primer extension and S1 nuclease protection assays. The excised fragment (gap sequence) was localized to expansion segment 5 within domain IV from which 86 bases are removed during the excision process. The gap region is flanked by the consensus sequence CGAAAG; however, comparison of expansion segment 5 sequences from T. spiralis, T. nativa, T. nelsoni and T. pseudospiralis, all of which undergo 'gap processing', demonstrates significant size and sequence heterogeneity and provides little evidence for additional consensus sequences which could be implicated in gap processing.

Animals↗

PubMed1987

Proto-oncogene c-fos is highly induced by disruption of neonatal but not of mature brain tissue.

The proto-oncogene c-fos can be induced in cells to high transcript levels by numerous exogenous stimuli. We show here by S1 nuclease protection assay that mere mechanical disaggregation and incubation at 37 degrees C induces high levels of c-fos hnRNA and subsequently of mature mRNA in neonatal mouse cerebellar tissue. This specific increase of c-fos steady-state levels is dependent on the incubation time with a maximal level of induction (over 40-fold) after approximately 1 h. The accumulation of c-fos transcripts is suppressed by alpha-amanitin while cycloheximide intensifies induction only moderately. Excessive elevation of c-fos mRNA levels is age-dependent and occurs only in early postnatal but not in adult cerebellar tissue. We conclude that the steady-state level of c-fos transcripts is inducible in a development-dependent manner, and thus may be involved in normal neurogenesis of the mammalian cerebellum.

Aging↗

PubMed1988

Expression of the gene for preproatriopeptin in the central nervous system of the rat.

Atriopeptin (AP) is a peptide hormone synthesized and secreted by the atria of the heart that participates in the regulation of fluid and electrolyte balance. AP-like materials have been detected immunologically in neurons in the central nervous system of the rat. In this study, we have used a solution hybridization-nuclease protection assay to determine whether the brain of the rat contains RNA coding preproatriopeptin, the atrial biosynthetic precursor of AP, and to study the regional distribution of preproatriopeptin mRNA in the brain. We have found that the brain contains mRNA identical to the atrial messenger RNA for preproatriopeptin. AP mRNA is differentially distributed in the brain; the highest concentration was found in the hypothalamus, followed by the cortex and septum, hippocampus, midbrain, spinal cord, olfactory bulb, striatum, and pons and medulla. Very low levels were found in the cerebellum, while no detectable AP mRNA was observed in retina, anterior pituitary, or rat liver. The presence of AP mRNA in the brain demonstrates that neurons have the capacity to synthesize preproatriopeptin identical to that produced in the heart. Bioactive peptides produced from this precursor may be endogenous central neuromodulators as well as a circulating hormone.

Animals↗

PubMed1988

Structural and functional analysis of the promoter region of the nerve growth factor gene.

Molecular clones containing the NGF gene promoter regions and exons I were isolated from mouse and rat genomic libraries with synthetic oligonucleotide hybridization probes that corresponded to the 5' end of mouse submandibular gland NGF mRNA. The nucleotide sequences of the 5' flanking regions and exons I were determined and compared. There was 95% similarity in exons I and the adjacent promoter regions between mouse and rat sequences. Further upstream, the similarity decreased to 76%. Both mouse and rat promoter regions were only 33% similar to the presumptive human NGF gene promoter region. Upstream from the capsite of submandibular gland NGF mRNA as determined by an S1-nuclease protection assay, two promoter-like TATA-boxes were found at positions -28 and -49, resp., and two CAAT-like boxes at -379 and -546, resp. Both promoter regions contained a cluster of conserved CpG dinucleotide sequences in a GC-rich island whereas less conserved upstream regions contained only one CpG sequence. The promoter regions were fused to the human growth hormone gene reporter function. Transient expression in L929 cells yielded appropriate fusion mRNAs and secretion of hGH, demonstrating that the cloned promoters are functional.

Animals↗

PubMed1989

Estradiol stimulates preoptic area-anterior hypothalamic proGnRH-GAP gene expression in ovariectomized rats.

The decapeptide gonadotropin-releasing hormone (GnRH) and the 56-amino acid GnRH-associated peptide (GAP) are derived from a common precursor translated from the proGnRH-GAP mRNA. Studies using solid-phase hybridization techniques (i.e., Northern blot analysis, dot blot analysis, or in situ hybridization autoradiography) have yielded a controversy as to whether estradiol stimulates, inhibits, or has any effect on proGnRH-GAP gene expression in the preoptic area-anterior hypothalamus (POA-AH) of the ovariectomized (OVX) rat. Using a sensitive and quantitative solution hybridization-nuclease protection assay, which ensures complete hybridization of target RNA to probe RNA, we examined the effects of OVX and estradiol replacement on the amount of proGnRH-GAP mRNA in individual POA-AH dissections. Rats sacrificed at different intervals after OVX showed a significant time-dependent decrease (34-60%) in the levels of POA-AH proGnRH-GAP mRNA relative to sham-operated animals; OVX rats treated with estradiol, however, had proGnRH-GAP mRNA levels comparable to those of sham-OVX animals. To verify these observations, levels of the proGnRH-GAP peptide, measured by radioimmunoassay with antibodies directed against the cleavage and amidation site between the GnRH and the GAP portions fo the precursor molecular, were also found to decrease (37%) after OVX and increase (63-85%) following estradiol replacement, relative to intact rats. These data support the view that estradiol stimulates the levels of both proGnRH-GAP mRNA and its primary translation product in the POA-AH region of the OVX rat.

Animals↗

PubMed1989

Intervening sequence-specific in situ hybridization: detection of the pro-opiomelanocortin gene primary transcript in individual neurons.

We describe here the in situ hybridization procedure which we have used to detect the pro-opiomelanocortin (POMC) gene primary transcript in nuclei of individual neurons in the periarcuate region of the hypothalamus. An exon-intron RNA probe was used to detect POMC primary transcript and mature mRNA in nuclear extracts of nucleic acids using a sensitive S1 nuclease protection assay. The levels per cell of nuclear primary transcript were similar to those seen in the anterior pituitary, suggesting that intervening sequence in situ hybridization should be feasible. A nonrepetitive complementary RNA probe specific for the first intervening sequence of the rat POMC gene (POMC IVS-A) was used to detect the POMC primary transcript in hypothalamic tissue sections by in situ hybridization. The distribution of nuclear localized autoradiographic grains was similar to that previously reported for immunocytochemically defined POMC neurons, suggesting that the procedure is also effective in brain cells.

Arcuate Nucleus of Hypothalamus↗

PubMed1990

Quantitative study of tyrosine hydroxylase mRNA in catecholaminergic neurons and adrenals during development and aging.

Using a solution hybridization-S1 nuclease protection assay, we quantitatively studied tyrosine hydroxylase mRNA (mRNATH) in catecholaminergic cells of the substantia nigra, hypothalamus, superior cervical ganglion, and adrenal of male rats from early neonatal life to old age. Throughout this time, the lowest level of mRNATH in any tissue was found in the youngest animals, and their development was associated with an increase in the quantity of mRNATH. However, the extent of the increase as well as the pattern of change was dependent on the tissue. The amount of mRNATH in the substantia nigra of 1-day-old pups was 162 +/- 7 attomoles (mean and S.E.M.), increasing to 877 +/- 39 amol at 14 days of age. Then, the amount fell to 480 +/- 25 amol at 6 weeks of age, but changed little between 6 weeks and 23 months of age. In the hypothalamus of 1-day-old pups, the quantity of mRNATH was 24 +/- 3 amol, increasing to 60 +/- 6 amol at 2 weeks and changed little thereafter. mRNATH in the superior cervical ganglion increased gradually until 10 months of age; at which time the amount was 3 times that of neonatal animals. In the adrenal, mRNATH increased continuously throughout the period of observation. The amount of mRNATH in the adrenal of 23-month-old animals was 25 times that in the adrenal of 4-day-old pups. These data suggest that tyrosine hydroxylase gene expression does not diminish in aged rats.

Adrenal Glands↗

PubMed1991

Alternative splicing of the beta A4 amyloid gene of Alzheimer's disease in cortex of control and Alzheimer's disease patients.

An S1 nuclease protection assay was designed to study the splicing pattern of the alternatively spliced beta A4 amyloid gene (APP gene) of Alzheimer's disease (AD). We determined the splicing pattern of the APP gene in fetal, adult, aged adult and AD human cortex. The results suggest that alternative splicing of the APP gene in AD is not significantly different from age-matched controls, but distinct from the developing fetal brain.

Adult↗