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Results for “Nuclease Protection Assays”

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PubMed2000

Up-regulation of novel intermediate filament proteins in primary fiber cells: an indicator of all vertebrate lens fiber differentiation?

The early embryonic development and expression patterns of the eye lens specific cytoskeletal proteins, CP49 and CP95, were determined for the chick and were found to be similar in both human and mouse. These proteins, as well as their homologs in other species, are obligate polymerization partners which form unique filamentous structures termed "beaded filaments." CP49 and CP95 appeared as protein products after 3 days of embryonic development in the chick during the elongation of primary fiber cells. Although limited data were obtained for human embryos at these early developmental timepoints, they were consistent with the interpretation that the up-regulation of these lens specific proteins began only after the initiation of lens vesicle closure. In situ hybridization with the mouse lens confirmed that message levels for beaded filament proteins were greatly elevated in differentiating primary fiber cells. Nuclease protection assays established that mRNA levels for CP49 remained relatively constant while CP95 mRNA levels increased once the process of secondary fiber formation was under way. Although present in relatively low abundance, the mRNA for a unique splice variant of CP49, CP49(INS), was also detected early in embryonic development and into adulthood. Peptide-specific antibodies directed against unique predicted sequences were able to confirm the protein expression of CP49(INS) in both embryonic and adult chick lens cells. These data present the first detailed study of the expression of CP49 and CP95 during early lens development. They suggest that the up-regulated expression of CP49 and CP95 could serve as pan-specific markers for all vertebrate lens fiber development.

Animals↗

PubMed1996

Lipopolysaccharide stimulates differential expression of myristoylated protein kinase C substrates in murine microglia.

Microglia rapidly respond to lipoplysaccharide (LPS) by transformation from resting to active states and secretion of several neuro- and immuno-regulators including tumour necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and interleukin 6 (IL-6). With longer LPS treatment, microglia are converted to reactive or phagocytic states with characteristics similar to macrophages in inflammation and injury processes. We have investigated LPS-mediated changes in two myristoylated substrates of protein kinase C (PKC): MARCKS (myristoylated alaninerich C kinase substrate) and MRP (MARCKS-related protein). Within 6 hours of addition, LPS induced a twofold increase in [3H]myristoylated and immunoreactive MARCKS protein and a sevenfold increase in MRP. The differential effect of LPS on expression of MRP vs. MARCKS was even more dramatic at the level of transcription: S1 nuclease protection assays revealed a 40-fold increase in MRP mRNA levels (maximum at 4-6 hours), whereas a threefold increase was observed for MARCKS. TNF alpha and colony-stimulating factor 1 (CSF-1), two cytokines which are induced by LPS, did not reproduce the observed effect of LPS on MARCKS and MRP gene transcription. CSF-1 also induced differential transcription of MRP, but of lower magnitude (threefold) and more sustained than by LPS. Accordingly, these two substrates for PKC are differentially up-regulated by LPS, apparently independent of TNF alpha or CSF-1.

Animals↗

PubMed2000

Transcriptional control elements and complex initiation pattern of the TATA-less bidirectional human thymidylate synthase promoter.

The nucleotide sequences that are important for transcription of the human thymidylate synthase gene were analyzed by deletion and site-directed mutagenesis of the promoter region. Deletion analyses from the 5' and 3' ends indicated the presence of multiple positive and negative elements. The promoter had approximately the same strength in the normal or inverted orientation. The region between 161 and 141 nt upstream of the translational start codon was found to be both necessary and sufficient for high-level promoter activity in both directions and was designated the essential promoter region. This region, which is highly conserved in human, mouse and rat TS promoters, contains potential binding sites for Ets, Sp1, and LSF transcription factors. Site directed mutagenesis of each of these elements led to large decreases in promoter strength. However, inactivation of potential Sp1 and E2F elements adjacent to the essential promoter region led to increases in promoter strength. The transcriptional start site pattern was analyzed by S1 nuclease protection assays of mRNA isolated from cells transiently transfected with TS minigenes. Multiple start sites were detected, most of which were between 160 and 120 nt upstream of the AUG codon.

Animals↗

PubMed1994

Gene therapy through signal transduction pathways and angiogenic growth factors as therapeutic targets in breast cancer.

Angiogenesis is a major new prognostic factor in breast cancer. Small vessels quantitatively assessed by staining with anti-CD31 antibodies correlate with lymph node involvement and are a better independent predictor of survival. There are many vascular growth factors, but predominant in primary tumors assessed by nuclease protection assays are vascular endothelial growth factor and platelet-derived endothelial cell growth factor. Acidic and basic fibroblast growth factor are also detectable. A common feature of these angiogenic factors is heparin binding, so novel analogues of suramin that can compete for heparin binding have been developed. These are more potent in vitro against endothelial cells and are less toxic in vivo, thereby giving a much better therapeutic ratio. Protein kinase C is also important in endothelial growth, as it is in carcinoma growth. Thus, a novel agent inhibiting this pathway, and inducing transforming growth factor-beta production has been assessed in a Phase I trial; this agent is bryostatin. It does not cause marrow suppression and has stimulatory effects of tumor necrosis factor-alpha and interleukin (IL)-6 production. High expression of epidermal growth factor (EGF) receptors and erbB-2 has been related to poor prognosis. EGF receptors are mainly regulated by transcription, as are some cases of high erbB-2 expression. Thus, a novel approach to gene therapy is being developed using direct tumor injection of cDNA, with a tumor specific promoter ligated to the IL-2 gene. This avoids many problems associated with targeting. Because IL-2 stimulation of cytotoxic T-cells will depend on appropriate antigen presentation, human lymphocyte antigen Class I expression was studied, as was the peptide transporter system RING4 (TAP1). Losses were found in 50% of cases, and in some cases only in lymph nodes but not primary cancers, thereby providing evidence for a role in suppressing metastasis. Thus, many new approaches to therapy are possible as a result of understanding growth factors and intracellular signaling pathways.

Angiogenesis Inducing Agents↗

PubMed1989

Fc gamma RII expression in resting and activated B lymphocytes.

In this report, we analyze the expression of the type II receptor for the Fc region of IgG (Fc gamma RII) in resting and lipopolysaccharide (LPS)-activated murine B lymphocytes. Fc gamma RII is encoded by two genes, alpha and beta. The beta gene encodes two mRNA, beta 1 and beta 2, which are generated by alternative splicing. Using an S1 nuclease protection assay, we found that resting and activated B lymphocytes express predominantly the beta 1 transcript. Very low levels of the beta 2 mRNA were detected in this assay, while no expression of the alpha transcript could be detected. Quantitative Northern blot analysis showed that the amount of Fc gamma RII beta mRNA was increased 9-fold in LPS-activated B lymphocytes. The expression of Fc gamma RII during the various phases of B cell activation was then studied by immunofluorescence using the monoclonal antibody 2.4G2. LPS stimulation induced an increase of the Fc gamma RII cellular pool as well as of its expression at the surface of B lymphocytes. The rise in Fc gamma RII surface expression occurred after the induction of class II antigens (Ia) and before transferrin receptor induction. Fc gamma RII expression was found to be enhanced during the G1 phase of the cell cycle since (a) only large cells (i.e. those that had entered the G1 phase) expressed an increased amount of Fc gamma RII and (b) blocking the entry of activated cells into the S phase (with the ion channel blocker quinine) did not affect the Fc gamma RII induction by LPS. Furthermore, only B cell activators that induced cells to enter into G1 [LPS and F(ab')2 anti-IgM antibodies, but not interleukin 4] caused an increase in the expression of Fc gamma RII. These results show that the increase in the membrane expression of Fc gamma RII occurs during the early G1 phase, establishing it as a marker for the entry of B lymphocytes into the cell cycle.

Animals↗

PubMed1994

Expression of neuronal src mRNA as a favorable marker and inverse correlation to N-myc gene amplification in human neuroblastomas.

Neuron-specific src mRNA, which is expressed in human brain tissue by alternative splicing, is associated with neural differentiation. Neuronal c-srcNI expression may be associated with the ability of neuroblastomas to mature; furthermore, c-srcN2 mRNA is induced in chemically differentiated neuroblastoma cells in vitro. The prognosis of a patient with a neuroblastoma is strongly affected by the ability of the tumor to differentiate in vivo. In order to clarify the relationship between neuronal src mRNA expression and the clinical outcome of a neuroblastoma, we analyzed the expression of src mRNA in neuroblastoma tissues from 28 patients by SI-nuclease-protection assay. N-myc gene amplification was also examined by Southern blot hybridization. The clinical significance of neuronal src mRNA expression and its relevance to N-myc gene amplification was also investigated. A high ratio (more than 10%) of c-srcN2 mRNA expression was observed in all early-stage tumors and in advanced neuroblastomas with a favorable prognosis. In contrast, in advanced neuroblastomas with an aggressive clinical phenotype, c-srcN2 mRNA expression was found at a low ratio (below 10%). Genomic amplification of the N-myc gene and expression of c-srcN2 mRNAs were inversely correlated. When combined with other prognostic markers such as N-myc gene amplification, the expression of c-srcN2 mRNA may be a new biological marker to predict the prognosis of patients with neuroblastomas.

Blotting, Southern↗

PubMed1985

Hormonal regulation of cell surface expression of the major histocompatibility antigen H-2Ld in transfected cells.

The murine major histocompatibility antigens are cell surface glycoproteins which play an important role in the recognition of foreign antigens by cytotoxic T lymphocytes. Modulation of the level of expression of histocompatibility antigens could therefore be useful for the study of the interaction between the antigen presenting cells and T lymphocytes. The glucocorticoid hormone-inducible promoter, located in the long terminal repeat of mouse mammary tumor virus, was used to replace the promoter region of a cloned H-2Ld class I gene. The chimeric gene was introduced into cultured cells. Glucocorticoid induction of MMTV LTR H-2Ld mRNA could be shown by blot analysis. An S1 nuclease protection assay indicated that the transfected cells accurately initiate the chimeric mRNA. Immunoprecipitation of H-2Ld protein with a specific monoclonal antibody showed inducibility also at the cellular protein level. Fluorescence-activated cell sorter analysis monitored a 3-fold increase of H-2Ld on the cell surface when the transfected cells were grown in the presence of dexamethasone. This increase of H-2Ld expression was accompanied by a corresponding decrease on the cell surface of the endogenous H-2Kk.

Animals↗

PubMed1986

Phytochrome-controlled expression of a wheat Cab gene in transgenic tobacco seedlings.

We have monitored changes in the chlorophyll a/b-binding protein (Cab) mRNA levels in etiolated wheat leaves exposed to light of different wavelengths by Northern blot hybridizations and 5' S1 nuclease protection assays. Accumulation of the Cab mRNA and the specific Cab-1 transcript is regulated by phytochrome. Transcript levels are elevated by red light and the red enhancement can be partially reversed by far-red light. Moreover, far-red light alone can elicit a small increase in the transcript levels. These characteristic features of the wheat Cab-1 phytochrome response can be recapitulated in etiolated seedlings of transgenic tobacco containing the Cab-1 gene. Analyses of a chimeric construct revealed that a 1.8-kb 5'-flanking fragment (-1816 to +31) of the Cab-1 gene can confer phytochrome response as well as leaf-specific expression on a heterologous coding sequence.

Blotting, Northern↗

PubMed1986

Primary structure and differential expression of glutamine synthetase genes in nodules, roots and leaves of Phaseolus vulgaris.

In plants, glutamine synthetase (GS) is the enzyme primarily responsible for the assimilation of ammonia into organic nitrogen. In Phaseolus vulgaris a number of isoenzymic forms of GS are found, each of which consists of eight subunits of mol. wt 41 000-45 000. The GS subunits of P. vulgaris have previously been shown to be encoded by a small multigene family and a partial cDNA clone for a nodule-specific GS subunit has been obtained. We report here the isolation and nucleotide sequencing of two essentially full-length GS cDNA clones (pR-1 and pR-2) from a root cDNA library and the deduced amino acid sequences of the corresponding GS subunits (355 amino acid residues each). The coding sequences of pR-1 and pR-2 are closely related (80% nucleotide homology, 88% amino acid homology), but their 5'- and 3'-untranslated regions have diverged almost completely. Both pR-1 and pR-2 are related to, but distinct from, the nodule GS clone, pcPvNGS-01 (or pN-1). Hybridization to genomic Southern blots showed that the three GS mRNAs are encoded by three seperate genes and indicated the existence of a fourth class of GS gene. An S1 nuclease protection assay demonstrated the presence of R-1 and R-2 mRNA in both roots and leaves and confirmed that expression of the N-1 gene is nodule-specific. Expression of the R-1 and R-2 genes in the roots did not change significantly during nodulation. However, only the R-1 gene is expressed in the nodules themselves, indicating that the R-2 gene is specifically repressed during nodule development.

Journal Article↗

PubMed1986

Proto-oncogene c-myc is expressed in cerebellar neurons at different developmental stages.

During post-natal cerebellar development the steady-state levels of c-myc transcripts exhibit characteristic changes. As determined by the S1 nuclease protection assay the level of c-myc transcript, which is very high in the late embryonic cerebellum, decreased to low levels shortly after birth. One week later there is a second period of c-myc mRNA accumulation followed by a marked decline to finally reach the low adult value. The second peak of high c-myc mRNA level correlates well with the proliferation of granule cell precursors, and it is characterized by a marked change in the ratio of the two types of transcripts started at the known c-myc promoters 1 and 2. This indicates a change in the cell population involved in the transcription of the c-myc gene. In situ hybridization shows transiently elevated c-myc mRNA levels in neurons of the cerebellar cortex. At post-natal days 3 and 10 (P3 and P10) c-myc transcripts are detectable in the superficial external granular layer composed primarily of mitotically active (neural precursor) cells. Purkinje cell somata show cytoplasmic label at P10. These large postmitotic neurons undergo rapid differentiation at this developmental stage. In the adult cerebellum the low c-myc mRNA level is apparently due to Purkinje cells with barely detectable amounts of c-myc transcripts. The vast majority of mature cerebellar neurons, the internal granule cells, have no specific hybridization signal for c-myc. We conclude that neurons in vivo can accumulate c-myc messenger during proliferation and/or differentiation, perhaps as a cellular response to an external signal.

Aging↗

PubMed1987

Site specific demethylation in the promoter of human gamma-globin gene does not alleviate methylation mediated suppression.

Human gamma A-globin genes with different patterns of methylation were synthesized in vitro. These methylated constructs were transfected into mouse L cells and their expression analyzed by S1-nuclease protection assays. We show that methylation downstream of +92 does not affect transcription while methylation downstream of -210 abolishes transcription. To test which of the six CpG dinucleotides between -210 and +92 plays a role in DNA methylation mediated suppression of transcription, we generated mutant gamma-globin genes which lack the three CpGs 5' to the cap site, the three CpGs 3' to the cap site, or all six CpGs. After in vitro methylation, we show that removal of methylated residues 5' or 3' to the cap site did not alleviate suppression, whereas removal of both 5' and 3' methylated residues restored partial expression. These results indicate that CpG methylation at different positions can cause suppression of expression and they are inconsistent with the hypothesis that site-specific demethylations in the promoter region can alleviate suppression. We propose that a minimum length of methylation-free DNA in the promoter area is the only requirement for expression of this promoter by ubiquitous transcription factors, and discuss these results with reference to methylation-free islands.

Animals↗

PubMed1989

Differential exon usage involving an unusual splicing mechanism generates at least eight types of NCAM cDNA in mouse brain.

The murine neural cell adhesion molecule (NCAM) is known to exist in three isoforms of different size, NCAM-180, -140 and -120 coded for by four transcripts of 6.9, 6.1, 4.8 and 2.7 kb in length. Since the differences between these isoforms are due to alternative splicing in the coding region for the transmembrane and cytoplasmic domains, the extracellular, N-terminal portion of NCAM seemed to be shared by all three protein forms. Here we report that the coding region for N-terminal domains of NCAM also contains at least two sites of alternative splicing, termed alpha and pi. Short additional sequences of 3, 18 and 30 nt in length can be introduced at these sites, which are located in the membrane-proximal 'stem' between the Ig-like domains and the membrane attachment site and within the Ig-like domain IV, respectively. Proof for at least eight different mRNAs has been found by sequencing and S1 nuclease protection assays of selected independent cDNA clones, and Northern blot analyses. If most combination of the splice patterns identified so far in mouse brain occurred, 24 different mRNAs could be generated coding for 18 different proteins. The shortest extra-sequence found inserted at splice site alpha consisted only of the trinucleotide AAG, raising questions about the mechanism of this particular insertion.

Amino Acid Sequence↗

PubMed1995

Chondrocytes isolated from mature articular cartilage retain the capacity to form functional gap junctions.

The distribution, expression, and functionality of gap junctions was examined in bovine chondrocytes (BCs) isolated from mature articular cartilage. BC cells displayed immunoreactivity for connexin 43 (Cx43), a specific gap junction protein. Cx43 protein expression was confirmed by Western blot analysis, and Cx43 mRNA was detected by nuclease protection assay. Additionally, BCs were shown to be functionally coupled, as revealed by dye transfer studies, and octanol, a gap junction uncoupler, greatly attenuated coupling. Furthermore, confocal microscopy of fluo-3 loaded BC cells revealed that deformation-induced cytosolic Ca2+ ion (Ca2+) signals propagated from cell-to-cell via gap junctions. To our knowledge, this is the first evidence suggesting that chondrocytes isolated from adult articular cartilage express functional gap junctions.

Animals↗

PubMed1989

Novel method for studying mRNA phenotypes in single or small numbers of cells.

Biological processes, such as growth control, are often governed by biochemical steps involving mRNA transcripts that are short-lived and have a low copy number. Furthermore, the cells involved in these processes are often available in low numbers from in vivo sources. We now report a method that is superior to in situ hybridization, RNA blot analysis, and the nuclease protection assay for the study of short-lived, low-copy-number mRNA transcripts. The method consists of a microprocedure for isolating RNA from one to a few thousand cells and two coupled enzymatic steps: reverse transcription of whole cellular RNA, followed by amplification of the cDNA by a specifically primed polymerase chain reaction to give specific cDNA fragments that can be visualized on agarose gels by ethidium bromide staining. With this method we have detected actin mRNA from a single cell, or less than 100 cRNA molecules, and have quantified differences in RNA concentrations of less than threefold. The reverse transcription reaction products can be divided for the polymerase chain reaction, and several mRNA species can be assayed simultaneously. Therefore, we call the method single-cell mRNA phenotyping. This technique is applicable to the analysis of low-copy-number growth factor transcripts in cells in culture and in vivo.

Actins↗

PubMed1995

In vivo occupancy of histone gene proximal promoter elements reflects gene copy number-dependent titratable transactivation factors and cross-species compatibility of regulatory sequences.

To assess systematically the structural and functional aspects of histone gene transcription within a chromosomal context, we stably integrated an extensive set of human histone H4 gene constructs into mouse C127 cells. Levels of expression were determined by S1 nuclease protection assays for multiple mouse monoclonal cell lines containing these human H4 genes. For each cell line, we quantitated the number of integrated human H4 genes by Southern blot analysis. The results indicate that the expression of the human H4 gene is in part copy number dependent at low gene dosages. However, the level of expression varies among different cell lines containing similar numbers of copies of the same H4 gene construct. This result suggests that position-dependent chromosomal integration effects contribute to H4 gene transcription, consistent with the roles of long-range gene organization and nuclear architecture in gene regulation. At high copy number, the level of human H4 gene expression per copy decreased, and endogenous mouse H4 mRNA levels were also reduced. Furthermore, in vivo occupancy at the human H4 gene immediate 5' regulatory elements, as defined by genomic fingerprinting, showed copy number-dependent protein/DNA interactions. Hence, human and mouse H4 genes compete for titratable transcription factors in a cellular environment. Taken together, these results indicate cross-species compatibility and suggest limited representation in vivo of the factors involved in regulating histone H4 gene transcription.

Animals↗

PubMed1986

Localization of human histone gene transcripts predominantly in the nonmatrix nuclear fraction.

Using S1 nuclease protection assays, we have examined the representation of cell cycle-dependent H4 histone RNAs in the nuclear matrix and nonmatrix nuclear fractions of human cells. Cytoplasmic and nuclear fractions were prepared from exponentially growing HeLa S3 cells by double detergent (sodium deoxycholate and NP40) lysis. The nuclear matrix and nonmatrix nuclear fractions were then prepared by digestion of nuclei with RNase-free DNase I and subsequent high-salt [0.4 M (NH4)2SO4] extraction. Subcellular fractions were characterized by 1) DNA, RNA, and protein composition; 2) electrophoretic analysis of the proteins in each fraction; 3) the representation of 45S ribosomal RNA precursors and processed 18S and 28S ribosomal RNAs; and 4) the presence of mitochondrial RNAs. In contrast to ribosomal and messenger RNA precursors, which are largely associated with the nuclear matrix, the human H4 histone RNAs in the nucleus were found predominantly in the nonmatrix nuclear fraction. The presence of H4 histone RNA in the nonmatrix nuclear fraction appeared to be coupled to DNA replication, since inhibition of DNA synthesis by hydroxyurea resulted in a loss of histone RNA from the nucleus. Our results suggest either that the association of histone RNAs with the nuclear matrix is very transient or that posttranscriptional modifications of the rapidly processed histone gene transcripts do not involve the nuclear matrix.

Cell Cycle↗

PubMed1989

Analysis of the multiple 5' and 3' termini of poly(A)+ and poly(A)-deficient thymidylate synthase mRNA in growth-stimulated mouse fibroblasts.

Thymidylate synthase (TS) mRNA content increases about 20-fold when growth-stimulated mouse cells progress from the G0/G1 phase into the S phase of the cell cycle. Previous studies, using a cell line in which the TS gene is amplified (LU3-7), indicated that transcriptional initiation as well as polyadenylation of the mRNA occur at several locations in unsynchronized cells. In the present study, we have used S1 nuclease protection assays to analyze the possible significance of the multiple transcriptional initiation and polyadenylation sites. We found that the same pattern of 5' and 3' termini were detected with RNA isolated from the overproducing cells as with RNA isolated from the parental mouse 3T6 cell line, demonstrating that the heterogeneous termini are not a consequence of gene amplification. There was no change in the pattern of 5' or 3' termini with either cell line during the progression from G1 phase through S phase in serum-stimulated cells. Therefore, the increase in TS mRNA content is not the result of differential utilization of the various transcriptional initiation or polyadenylation sites. Analyses of poly(A)- deficient cytoplasmic TS RNA showed that the 5' termini were the same as those found in poly(A) + mRNA. However, the 3' termini were extremely heterogeneous in length. Although some of the poly(A)- deficient RNA extended beyond the normal site of polyadenylation, most of it was shorter than full-length TS mRNA.

Animals↗

PubMed1994

Human interleukin 1 beta: corticotropin releasing factor and ACTH release and gene expression in the male rat: in vivo and in vitro studies.

Numerous studies have shown that interleukin 1 (IL1), a cytokine secreted by macrophages, is capable of stimulating the hypothalamo-pituitary-adrenal (HPA) axis. Nevertheless, the sites involved in IL1 stimulation of the HPA axis remain, to date, subjects of controversy. In the present study, using in vivo and in vitro approaches, we tried to characterize the route by which IL1 acts on the HPA axis. In vivo, after an i.p. injection of human IL1 beta (1 microgram/rat), we measured plasma ACTH concentration, anterior pituitary (AP) ACTH content, hypothalamic (HT) corticotropin releasing factor (CRF) content, and also AP pro-opiomelanocortin (POMC) and HT CRF gene expression. ACTH and CRF were measured by specific radioimmunoassays (RIAs), and solution hybridization nuclease protection assay was used for quantification of nuclear POMC precursor RNA and nuclear and cytoplasmic POMC and CRF mRNA. Human IL1 beta provoked an increase in ACTH plasma concentration, a decrease in AP ACTH content, and a prolonged increase in AP POMC primary transcript levels (around 100%). A significant increase in AP POMC primary transcript content was evident 30 min after injection of hIL1 beta, while cytoplasmic POMC mRNA levels were increased in the AP only at 4 hr after injection of hIL1 beta. We did not observe an effect of hIL1 beta on either HT CRF content or HT CRF cytoplasmic mRNA levels. In order to characterize a possible direct effect of hIL1 beta at the AP level, we used an AP perifusion system to analyse the effect of hIL1 beta and CRH on ACTH release and on POMC gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗