scholarly journals IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION

1974 ◽  
Vol 63 (2) ◽  
pp. 414-419 ◽  
Author(s):  
D. Conkie ◽  
N. Affara ◽  
P. R. Harrison ◽  
J. Paul ◽  
K. Jones
Keyword(s):  
Messenger Rna ◽  
Mrna Levels ◽  
Globin Mrna ◽  
Mrna Accumulation ◽  
True Solution ◽  
Cytoplasmic Rna ◽  
Friend Cells ◽  
Experimental Approaches ◽  
Mouse Cells

Globin messenger RNA (mRNA) levels in Friend virus-transformed mouse cells have been estimated by in situ hybridization of DNA copy (cDNA) to fixed preparations of cells and by hybridization of cDNA to extracted cytoplasmic RNA in true solution. The results obtained by both methods agree in showing that a low level of globin mRNA can be detected in untreated Friend cells. The levels of hemoglobin and globin mRNA have also been correlated after treatment of Friend cells with dimethyl sulfoxide (DMSO). The results obtained by both experimental approaches show that there is a minimum period of treatment with DMSO required in order that Friend cells may become hemoglobinized, and that this period coincides with the time when globin mRNA accumulates. Moreover, bromodeoxyuridine prevents both hemoglobin and globin mRNA accumulation.

scholarly journals IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION

1974 ◽  
Vol 63 (2) ◽  
pp. 402-413 ◽  
Author(s):  
P. R. Harrison ◽  
D. Conkie ◽  
N. Affara ◽  
J. Paul
Keyword(s):  
In Situ Hybridization ◽  
Messenger Rna ◽  
Fetal Liver ◽  
Liver Cells ◽  
Mrna Levels ◽  
Messenger Rnas ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Fetal Liver Cells

Globin mRNA levels in 11–15-day mouse fetal liver cells have been estimated by in situ hybridization of a highly labeled DNA copy (cDNA) of adult globin messenger RNAs (mRNAs) (globin cDNA) to fixed preparations of cells. Under the conditions employed, no significant in situ hybridization occurred to lymphoma cells (L 51787), mouse L cells, or hepatocytes; whereas reticulocytes from phenyl hydrazine-treated mice showed extensive in situ hybridization. The proportion of fetal liver cells showing predominantly cytoplasmic in situ hybridization increased from about 30% at the 11th day of development to 80–85% by days 13–15. Unlike more mature cells, proerythroblasts did not show in situ hybridization, except to a slight extent at later stages of development. These studies therefore indicate that globin mRNAs begin to accumulate during or shortly after the proerythroblastbasophilic erythroblast transition. The fact that certain immature erythroid cells from 14-day fetal liver contain substantial amounts of globin mRNAs has been confirmed by comparing the hybridization in solution of globin cDNA to cytoplasmic RNA extracted from total fetal liver cells or from immature erythroid cells obtained by treatment of fetal liver cells with an antiserum raised against erythrocytes.

Production of mouse globin in heterokaryons of mouse erythroleukaemia cells and human fibroblasts

1977 ◽  
Vol 26 (1) ◽  
pp. 347-357
Author(s):  
B.P. Alter ◽  
S.C. Goff ◽  
D.G. Hillman ◽  
A.B. Deisseroth ◽  
B.G. Forget
Keyword(s):  
Amino Acids ◽  
Human Fibroblasts ◽  
Fusion System ◽  
Globin Genes ◽  
Polyacrylamide Gels ◽  
Globin Mrna ◽  
Globin Chains ◽  
Cytoplasmic Rna ◽  
Friend Cells ◽  
Globin Synthesis

In an effort to activate the globin genes of non-erythroid cells, tetraploid murine erythroleukaemia cells (Friend cells) were fused with diploid human amniotic fibroblasts. When the Friend cells were pretreated with dimethylsulphoxide, an average of 27% heterokaryons was observed. These cells stained with benzidine, an indication that they contained haemoglobin. The cells incorporated radioactive amino acids into proteins. Electrophoresis of [3H]leucine-labelled lysates on SDS urea polyacrylamide gels indicated that up to 7% of the newly synthesized protein co-electrophoresed with globin. CM cellulose chromatography demonstrated the presence of mouse but not human globin chains. Hybridization analyses of cytoplasmic RNA also revealed only mouse globin mRNA in the heterokaryons. Although heterokaryons form readily between mouse erythroleukaemia cells and human fibroblasts, and globin synthesis does occur, only the erythroid partner in the fusion system employed here directs globin production.

Control of proliferin gene expression in serum-stimulated mouse cells

1987 ◽  
Vol 7 (6) ◽  
pp. 2080-2086
Author(s):  
D I Linzer ◽  
E L Wilder
Keyword(s):  
Protein Synthesis ◽  
S Phase ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Serum Stimulation ◽  
Posttranscriptional Processing ◽  
A Cell ◽  
Mouse Cells ◽  
Stimulation Of

The serum-inducible expression of proliferin genes in BALB/c 3T3 cells was found to be dependent on both protein synthesis and an extended presence of serum in the medium. Even though no mature proliferin mRNA was detected in serum-starved cells, transcription of the proliferin genes occurred in these resting-cell cultures, indicating that posttranscriptional events may be important for regulating proliferin mRNA levels. These results suggest that protein synthesis after serum stimulation of quiescent mouse fibroblasts is required for posttranscriptional processing or stabilization of proliferin RNA. Proliferin RNA levels were found to be heterogeneous among serum-stimulated cells analyzed by in situ hybridization. This heterogeneity is probably due to asynchrony in the population and may point to a correlation between the time of proliferin expression and the time of entry of a cell into S phase.

scholarly journals Control of proliferin gene expression in serum-stimulated mouse cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2080-2086 ◽  
Author(s):  
D I Linzer ◽  
E L Wilder
Keyword(s):  
Protein Synthesis ◽  
S Phase ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Serum Stimulation ◽  
Posttranscriptional Processing ◽  
A Cell ◽  
Mouse Cells ◽  
Stimulation Of

The serum-inducible expression of proliferin genes in BALB/c 3T3 cells was found to be dependent on both protein synthesis and an extended presence of serum in the medium. Even though no mature proliferin mRNA was detected in serum-starved cells, transcription of the proliferin genes occurred in these resting-cell cultures, indicating that posttranscriptional events may be important for regulating proliferin mRNA levels. These results suggest that protein synthesis after serum stimulation of quiescent mouse fibroblasts is required for posttranscriptional processing or stabilization of proliferin RNA. Proliferin RNA levels were found to be heterogeneous among serum-stimulated cells analyzed by in situ hybridization. This heterogeneity is probably due to asynchrony in the population and may point to a correlation between the time of proliferin expression and the time of entry of a cell into S phase.

scholarly journals Regulation of tyrosine hydroxylase mRNA in catecholaminergic cells of embryonic rat: analysis by in situ hybridization.

1989 ◽  
Vol 37 (1) ◽  
pp. 1-5 ◽  
Author(s):  
G M Jonakait ◽  
M Rosenthal ◽  
J I Morrell
Keyword(s):  
In Situ Hybridization ◽  
Tyrosine Hydroxylase ◽  
Messenger Rna ◽  
Sympathetic Ganglia ◽  
Mrna Levels ◽  
Pregnant Rats ◽  
Reserpine Treatment ◽  
Catecholaminergic Cells ◽  
Th Mrna

In situ hybridization was used to examine the appearance of mRNA specific for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine (CA) biosynthesis, in neural crest derivatives of the rat embryo. These derivatives include sympathetic ganglia and transient catecholaminergic cells of embryonic intestine. Messenger RNA is first detected in sympathetic ganglia at E11.5, the age corresponding to the initial immunocytochemical expression of TH protein. In older embryos increased accumulation of TH-specific mRNA in sympathetic ganglia parallels the increase in TH immunoreactivity. By contrast, mRNA for TH is difficult to detect in embryonic intestines at E11.5 but is found instead in cells clustered at the dorsal boundaries of the pharynx and foregut. Cells expressing TH mRNA are infrequently found in embryonic intestines at any age, even though TH protein is immunohistochemically apparent. Treatment of pregnant rats with doses of reserpine, known to increase circulating levels of glucocorticoid hormones and prolong the expression of TH protein in embryonic gut cells, dramatically but transiently increases the number of gut cells at E12.5 with detectable TH mRNA. After E13.5 TH mRNA is undetectable even in reserpine-treated guts. Reserpine treatment also increases the labeling density in sympathetic ganglia. Taken together, these data are consistent with the hypothesis that the microenvironment of the embryonic intestine affects gene expression directly to alter phenotype. Moreover, although reserpine administration briefly increases TH mRNA levels, the effect is short-lived and does not alter neurotransmitter phenotypic conversion.

scholarly journals Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.

1980 ◽  
Vol 87 (1) ◽  
pp. 47-54 ◽  
Author(s):  
T Pederson ◽  
N G Davis
Keyword(s):  
Nuclear Structure ◽  
Messenger Rna ◽  
Globin Gene ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Ribonucleoprotein Particles ◽  
Particle Form ◽  
Nuclear Rna ◽  
Friend Cells

To explore the relationships between transcription, messenger RNA (mRNA) processing, and nuclear structure, ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNP) have been purified from globin-producing mouse Friend erythroleukemia cells. These nuclear hnRNP particles sediment at 50S-200S and contain, in addition to high molecular weight hnRNA, a specific set of nuclear proteins predominated by a major component of approximately 38,000 mol wt. The hnRNP particles are free of histones and ribosomal structural proteins, indicating their purification from the two other major nucleoprotein components of the nucleus: chromatin and nucleolar ribosomal precursor RNP particles. Th authenticity of the Friend cell hnRNP particles is demonstrated by the results of reconstruction experiments with deproteinized hnRNA, and by the resistance of the articles to dissociation during isopycnic banding in Cs2SO4 gradients without prior aldehyde fixation. Hybridization analysis with cloned mouse beta-globin DNA demonstrates that hnRNP particles from induced Friend cells contain newly synthesized transcripts of the beta-globin gene. Agarose gel electrophoresis of hnRNP particle-derived RNA denatured in glyoxal followed by "Northern" transfer to diazobenzyloxymethyl paper and hybridization with 32P-labeled cloned mouse beta-globin DNA reveals the presence in hnRNP of two size classes of beta-globin gene transcripts, the larger of which corresponds to the pre-spliced 15S beta-globin mRNA precursor previously identified in whole nuclear RNA, and the smaller of which corresponds to completely processed 9S beta-globin mRNA. These results establish, for the first time, that the nuclear transcripts of a specific, well-defined eukaryotic structural gene can be isolated in an RNP particle form, and that their RNP structure persists throughout mRNA splicing.

scholarly journals The Serum- and Glucocorticoid-Induced Kinase Is a Physiological Mediator of Aldosterone Action*

Endocrinology ◽  
2001 ◽  
Vol 142 (4) ◽  
pp. 1587-1594 ◽  
Author(s):  
Aditi Bhargava ◽  
Meryl J. Fullerton ◽  
Kathy Myles ◽  
Timothy M. Purdy ◽  
John W. Funder ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Messenger Rna ◽  
Time Course ◽  
Rat Kidney ◽  
Distal Colon ◽  
Mrna Levels ◽  
Epithelial Tissues ◽  
Sodium And Potassium

Abstract Aldosterone plays a major role in regulating sodium and potassium flux in epithelial tissues such as kidney and colon. Recent evidence suggests that serum- and glucocorticoid-regulated kinase (SGK) is induced by aldosterone and acts as a key mediator of aldosterone action in epithelial tissues. Induction of SGK messenger RNA (mRNA) has previously been shown within 30 min of addition of supraphysiological doses of aldosterone to Xenopus A6 cells and within 4 h in rat kidney in vivo. In this study we determined the time course of SGK induction, at doses of aldosterone in the physiological range, in rat kidney and colon, using Northern and Western blot analyses and in situ hybridization and determined concurrent changes in urinary sodium and potassium excretion by Kagawa bioassay. On Northern blot analysis, SGK mRNA levels were significantly elevated in both kidney and colon 60 min after the injection of aldosterone. SGK protein in late distal colon was significantly elevated 2 and 4 h after aldosterone treatment. In situ hybridization showed SGK mRNA to be induced in renal collecting ducts and distal tubular elements in both cortex and medulla by doses of aldosterone of 0.1 μg/100 g BW or more within 30 min of steroid treatment. Significant changes in urinary composition were similarly seen with an aldosterone dose of 0.1 μg/100 g BW from 90 min after aldosterone injection. The early onset of SGK induction in kidney and colon and the correlation with urinary changes in terms of both time course and dose response suggest that SGK plays an important role in mediating the effects of aldosterone on sodium homeostasis in vivo.

scholarly journals Regulation of beta-globin mRNA accumulation by heme in dimethyl sulfoxide (DMSO)-sensitive and DMSO-resistant murine erythroleukemia cells

Blood ◽  
1994 ◽  
Vol 83 (6) ◽  
pp. 1662-1667 ◽  
Author(s):  
Y Fukuda ◽  
H Fujita ◽  
L Garbaczewski ◽  
S Sassa
Keyword(s):  
Dimethyl Sulfoxide ◽  
Erythroid Differentiation ◽  
Mrna Levels ◽  
Beta Globin ◽  
Globin Mrna ◽  
Mrna Accumulation ◽  
Erythroleukemia Cells ◽  
Dmso Treatment ◽  
Free Heme ◽  
Murine Erythroleukemia

The level of mRNA encoding beta-globin was examined in dimethyl sulfoxide (DMSO)-sensitive (DS), and DMSO-resistant (DR) murine erythroleukemia (MEL) cells. DR cells lack erythroid-specific delta- aminolevulinate (ALA) synthase (AL-AS-E), and fail to undergo erythroid differentiation following treatment with DMSO. Treatment of cells with DMSO markedly increased ALAS-E mRNA in DS cells, while the same treatment downregulated the nonspecific ALA synthase (ALAS-N) mRNA levels in both DS and DR cells. The levels of beta-globin mRNA, heme content, and hemoglobin in DS cells increased, while those in DR cells decreased following treatment with DMSO. Treatment of DR cells with hemin caused an increase in beta-globin mRNA and hemoglobin, and partially restored the DMSO-mediated suppression of beta-globin mRNA and hemoglobin synthesis. DMSO treatment decreased heme oxygenase (HO) mRNA in hemin-treated DS cells, but not in hemin-treated DR cells. These findings indicate that heme is necessary for accumulation of the beta-globin transcript during erythroid differentiation, and that hemin- mediated HO induction becomes markedly downregulated in differentiated erythroid cells, presumably because less free heme is available for HO induction by a greater demand for the synthesis of hemoglobin.

Differential dopamine-induced prolactin mRNA levels in various prolactin-secreting cell (sub)populations

1992 ◽  
Vol 132 (3) ◽  
pp. 401-409 ◽  
Author(s):  
M. Kazemzadeh ◽  
B. Velkeniers ◽  
P. Herregodts ◽  
R. Collumbien ◽  
E. Finné ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Pituitary Cells ◽  
Low Density ◽  
Mrna Levels ◽  
Percoll Gradient ◽  
Secreting Cell ◽  
Mrna Accumulation ◽  
Cell Layers ◽  
Prolactin Mrna

ABSTRACT We have examined the effects of dopamine on prolactin gene expression using quantitative in-situ hybridization histochemistry in different pituitary cell (sub)populations separated according to their density on a discontinuous Percoll gradient. Administration of dopamine resulted in a drastic reduction in hybridization of 35S-labelled DNA probe complementary to prolactin mRNA in total pituitary cells and in lactotrophs with low density. In contrast, dopamine significantly stimulated mRNA accumulation in prolactin-secreting cells with high density compared with other cell layers. The combined use of Percoll gradient and quantitative in-situ hybridization is a valuable and sensitive method with which to examine prolactin-secreting cell response to a given stimulation. Prolactin-secreting cells with high and low density clearly show functional heterogeneity in their response to dopamine. Journal of Endocrinology (1992) 132, 401–409

Pro-opiomelanocortin messenger RNA levels increase in the fetal sheep pituitary during late gestation

1991 ◽  
Vol 131 (3) ◽  
pp. 483-489 ◽  
Author(s):  
K. Yang ◽  
J. R. G. Challis ◽  
V. K. M. Han ◽  
G. L. Hammond
Keyword(s):  
Messenger Rna ◽  
Blot Hybridization ◽  
Late Gestation ◽  
Northern Blot Hybridization ◽  
Mrna Levels ◽  
Plasma Acth ◽  
Mrna Accumulation ◽  
Fetal Sheep ◽  
Fetal Plasma ◽  
Pomc Mrna

ABSTRACT Plasma levels of ACTH and cortisol in fetal sheep increase progressively during late pregnancy, providing the stimulus for birth. However, little information is available concerning either sources of pro-opiomelanocortin (POMC, the precursor to ACTH) or changes in POMC gene expression, which may be responsible for the elevated fetal plasma ACTH concentrations. We therefore studied the relative amount of POMC mRNA in fetal sheep hypothalami, anterior pituitaries and adrenals at discrete times of pregnancy between day 60 and term (approximately 145 days) and from newborn lambs. Total RNA from these tissues was analysed by Northern blot hybridization using a human POMC DNA probe, and the amount of POMC mRNA was expressed relative to the signal obtained for 18S ribosomal RNA. A single 1·2 kb transcript was detected by day 60 in the anterior pituitary, and its relative amount did not change significantly until after days 125–130. Pituitary POMC mRNA levels increased significantly at days 138–143, remained elevated at term and increased further in newborn lambs. In contrast, POMC mRNA was undetectable in hypothalami and adrenal glands of fetuses at all ages. The results suggested that the prepartum rise in plasma ACTH concentrations in fetal sheep is due to increased POMC biosynthesis in the fetal pituitary. The increase in POMC mRNA occurs at a time when fetal plasma cortisol concentrations are elevated, indicating that the negative feedback effects of circulating glucocorticoids on the fetal hypothalamicpituitary axis may be obscured by other mechanisms that increase pituitary POMC mRNA accumulation during the last week of gestation. Journal of Endocrinology (1991) 131, 483–489

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