A cell cycle analysis of growth-related genes expressed during T lymphocyte maturation.

Fetal liver or bone marrow-derived T lymphocyte precursors undergo extensive, developmentally regulated proliferation in response to inductive signals from the thymic microenvironment. We have used neonatal mouse thymocytes size-separated by centrifugal elutriation to study the cell cycle stage-specific expression of several genes associated with cell proliferation. These include genes involved in the biosynthesis of deoxyribonucleotide precursors, such as dihydrofolate reductase (DHFR), thymidylate synthase (TS), and the M1 and M2 subunits of ribonucleotide reductase, as well as c-myc, a cellular oncogene of unknown function. Using nuclear run-on assays, we observed that the transcription rates for these genes, with the exception of TS, are essentially invariant not only throughout the cell cycle in proliferating cells, but also in noncycling (G0) cells. The TS gene showed a transient increase in transcription rate in cells which bordered between a proliferating and nonproliferating status. Studies of an elutriated T cell line, S49.1, yielded similar results, indicating that the process of immortalization has not affected the transcriptional regulation of these genes. Analysis of steady-state mRNA levels using an RNase protection assay demonstrated that the levels of DHFR and TS mRNA accumulate as thymocytes progress through the cell cycle. In contrast, only the M2 subunit of ribonucleotide reductase showed cyclic regulation. Finally, in contrast to cultured cell models, we observed an abrupt fivefold increase in the steady-state level of c-myc mRNA in the transition from G1 to S-phase. We conclude from these studies that the transcriptional regulation of specific genes necessary for cellular proliferation is a minor component of the developmental modulation of the thymocyte cell cycle.

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Corticosteroid receptor mRNA expression is unaffected by corticosteroids in rat kidney, heart, and colon

AJP Cell Physiology ◽

10.1152/ajpcell.1996.270.5.c1343 ◽

1996 ◽

Vol 270 (5) ◽

pp. C1343-C1353 ◽

Cited By ~ 27

Author(s):

B. Escoubet ◽

C. Coureau ◽

M. Blot-Chabaud ◽

J. P. Bonvalet ◽

N. Farman

Keyword(s):

Steady State ◽

Rat Kidney ◽

Hormone Replacement ◽

Mrna Level ◽

Rnase Protection Assay ◽

Distal Colon ◽

Mrna Levels ◽

Adult Rats ◽

Rnase Protection ◽

Steady State Levels

Hormones can regulate the expression of their own receptor. We have examined whether adrenalectomy (ADX) and hormone replacement by physiological doses of aldosterone or dexamethasone could modulate the expression of glucocorticoid receptor (GR) or mineralocorticoid receptor (MR) at the mRNA level in the rat kidney, distal colon, and heart. Adult rats were adrenalectomized and received or did not receive an infusion of aldosterone (5 micrograms.100 g-1.day-1) or dexamethasone (10 micrograms.100 g-1.day-1). No significant change in steady-state levels of both MR and GR mRNA was detectable by using ribonuclease (RNase) protection assay (RPA) after either ADX or hormone replacement. Because the kidney is heterogeneous with regard to MR expression, RPA was adapted for measurements on microdissected nephron segments. GR mRNA is expressed at comparable levels all along the nephron, whereas MR mRNA is restricted to the distal nephron. No effect of ADX or GR and MR mRNA levels was detected in any nephron segment that was either aldosterone sensitive or insensitive. In situ hybridization confirmed the absence of corticosteroid-dependent modulation of MR mRNA in all kidney cell types. We conclude that variations of corticosteroid status do not affect MR and GR mRNA steady-state levels in heart, colon, and kidney and thus do not participate to the functional adaptations that are known to depend on hormonal status.

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Effect of growth hormone administration on IGF binding protein-3 mRNA levels in porcine tissues

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0220261 ◽

1999 ◽

Vol 22 (3) ◽

pp. 261-272 ◽

Cited By ~ 6

Author(s):

V Dunaiski ◽

FR Dunshea ◽

PE Walton ◽

C Goddard

Keyword(s):

Steady State ◽

Binding Protein ◽

Rnase Protection Assay ◽

Mrna Levels ◽

Short Term ◽

Gh Treatment ◽

Rnase Protection ◽

Igf I ◽

Class 1 ◽

Igfbp 3

The effect of short-term GH treatment on steady-state insulin-like growth factor binding protein-3 (IGFBP-3) mRNA levels in liver, kidney, longissimus dorsi muscle, stomach and jejunum was examined in pigs. Ten female crossbred pigs were allocated to either saline or GH (70 microg/kg/day) treatment by subcutaneous injection for 4 days. They were allowed to feed ad libitum, and were weighed daily. At the end of the treatment period, the pigs were slaughtered and samples of liver, kidney, skeletal muscle, stomach and jejunum were collected and total RNA was extracted. Steady-state levels of IGFBP-3 mRNA were quantified by RNase protection assay and were compared with the level of IGF-I class 1 and class 2 transcripts. IGFBP-3 mRNA increased in response to GH in both liver and kidney, but not in the other tissues sampled. Hepatic IGF-I mRNA responded to short-term GH treatment with a fourfold increase in IGF-I class 1 mRNA and an eightfold increase in IGF-I class 2 mRNA, which was liver specific. IGF-I class 1 mRNA was not responsive to GH treatment in other tissues. The short-term nature of this treatment suggests that the increase in hepatic IGFBP-3 and IGF-I transcripts is a relatively early response to treatment with GH, and that the increase in plasma concentrations of IGFBP-3 in response to GH are derived from the liver, the kidney, or both.

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Role of passive and adaptive immunity in influencing enterocyte-specific gene expression

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00130.2003 ◽

2003 ◽

Vol 285 (4) ◽

pp. G714-G725 ◽

Cited By ~ 26

Author(s):

Shannon L. Jenkins ◽

Jiafang Wang ◽

Mukta Vazir ◽

Jose Vela ◽

Omar Sahagun ◽

...

Keyword(s):

Gene Expression ◽

Small Intestine ◽

Steady State ◽

Rnase Protection Assay ◽

Specific Gene ◽

Mrna Levels ◽

Developmentally Regulated ◽

Rnase Protection ◽

Distal Small Intestine ◽

Steady State Levels

Numerous genes expressed by intestinal epithelial cells are developmentally regulated, and the influence that adaptive (AI) and passive (PI) immunity have in controlling their expression has not been evaluated. In this study, we tested the hypothesis that both PI and AI influenced enterocyte gene expression by developing a breeding scheme that used T and B cell-deficient recombination-activating gene ( RAG) mice. RNA was isolated from the liver and proximal/distal small intestine at various ages, and the steady-state levels of six different transcripts were evaluated by RNase protection assay. In wild-type (WT) pups, all transcripts [Fc receptor of the neonate ( FcRn), polymeric IgA receptor ( pIgR), GLUT5, lactase-phlorizin hydrolase ( lactase), apical sodium-dependent bile acid transporter ( ASBT), and Na+/glucose cotransporter ( SGLT1)] studied were developmentally regulated at the time of weaning, and all transcripts except ASBT had the highest levels of expression in the proximal small intestine. In WT suckling pups reared in the absence of PI, pIgR mRNA levels were increased 100% during the early phase of development. In mice lacking AI, the expression of pIgR and lactase were significantly attenuated, whereas FcRn and GLUT5 levels were higher compared with WT mice. Finally, in the absence of both passive and active immunity, expression levels of pIgR and lactase were significantly lower than similarly aged WT mice. In summary, we report that the adaptive and passive immune status of mice influences steady-state mRNA levels of several important, developmentally regulated enterocyte genes during the suckling and weaning periods of life.

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Genotype-dependent and non-gradient patterns of RSV gene expression

10.1101/641878 ◽

2019 ◽

Author(s):

Felipe-Andrés Piedra ◽

Xueting Qiu ◽

Michael N. Teng ◽

Vasanthi Avadhanula ◽

Annette A. Machado ◽

...

Keyword(s):

Gene Expression ◽

Transcriptional Regulation ◽

Steady State ◽

Transcription Initiation ◽

Mrna Levels ◽

Viral Pathogen ◽

Negative Strand ◽

Gene Start ◽

Syncytial Virus ◽

Conserved Gene

AbstractRespiratory syncytial virus (RSV) is a nonsegmented negative-strand (NNS) RNA virus and a leading cause of severe lower respiratory tract illness in infants and the elderly. Transcription of the ten RSV genes proceeds sequentially from the 3’ promoter and requires conserved gene start (GS) and gene end (GE) signals. Previous studies using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of gene transcription. However, recent reports show data that appear inconsistent with a gradient. To better understand RSV transcriptional regulation, mRNA abundances from five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines and cotton rats infected with virus isolates belonging to four different genotypes (GA1, ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours post-infection. Steady-state patterns were genotype-specific and non-gradient, where mRNA levels from the G (attachment) gene exceeded those from the more promoter-proximal N (nucleocapsid) gene across isolates. Transcript stabilities could not account for the non-gradient patterns observed, indicating that relative mRNA levels more strongly reflect transcription than decay. While the GS signal sequences were highly conserved, their alignment with N protein in the helical ribonucleocapsid, i.e., N-phase, was variable, suggesting polymerase recognition of GS signal conformation affects transcription initiation. The effect of GS N-phase on transcription efficiency was tested using dicistronic minigenomes. Ratios of minigenome gene expression showed a switch-like dependence on N-phase with a period of seven nucleotides. Our results indicate that RSV gene expression is in part sculpted by polymerases that initiate transcription with a probability dependent on GS signal N-phase.Author SummaryRSV is a major viral pathogen that causes significant morbidity and mortality, especially in young children. Shortly after RSV enters a host cell, transcription from its nonsegmented negative-strand (NNS) RNA genome starts at the 3’ promoter and proceeds sequentially. Transcriptional attenuation is thought to occur at each gene junction, resulting in a gradient of gene expression. However, recent studies showing non-gradient levels of RSV mRNA suggest that transcriptional regulation may have additional mechanisms. We show using RSV isolates belonging to four different genotypes that gene expression is genotype-dependent and one gene (the G or attachment gene) is consistently more highly expressed than an upstream neighbor. We hypothesize that variable alignment of highly conserved gene start (GS) signals with nucleoprotein (i.e., variable GS N-phase) can affect transcription and give rise to non-gradient patterns of gene expression. We show using dicistronic RSV minigenomes wherein the reporter genes differ only in the N-phase of one GS signal that GS N-phase affects gene expression. Our results suggest the existence of a novel mechanism of transcriptional regulation that might play a role in other NNS RNA viruses.

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Integrin alpha subunit mRNAs are differentially expressed in early Xenopus embryos

Development ◽

10.1242/dev.117.4.1239 ◽

1993 ◽

Vol 117 (4) ◽

pp. 1239-1249 ◽

Cited By ~ 7

Author(s):

C.A. Whittaker ◽

D.W. DeSimone

Keyword(s):

In Situ Hybridization ◽

Rnase Protection Assay ◽

Early Embryo ◽

Mrna Levels ◽

Rnase Protection ◽

Dorsal Midline ◽

Transmembrane Receptors ◽

Early Embryos ◽

Whole Mount

Adhesion of cells to extracellular matrix proteins is mediated, in large part, by transmembrane receptors of the integrin family. The identification of specific integrins expressed in early embryos is an important first step to understanding the roles of these receptors in developmental processes. We have used polymerase chain reaction methods and degenerate oligodeoxynucleotide primers to identify and clone Xenopus integrin alpha subunits from neurula-stage (stage 17) cDNA. Partial cDNAs encoding integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and an alpha IIb-related subunit were cloned and used to investigate integrin mRNA expression in early embryos by RNase protection assay and whole-mount in situ hybridization methods. Considerable integrin diversity is apparent early in development with integrins alpha 2, alpha 3, alpha 4, alpha 5 and alpha 6 each expressed by the end of gastrulation. Both alpha 3 and alpha 5 are expressed as maternal mRNAs. Zygotic expression of alpha 2, alpha 3, alpha 4 and alpha 6 transcripts begins during gastrulation. Integrin alpha 5 is expressed at relatively high levels during cleavage, blastula and gastrula stages suggesting that it may represent the major integrin expressed in the early embryo. We demonstrated previously that integrin beta 1 protein synthesis remains constant following induction of stage 8 animal cap cells with activin (Smith, J. C., Symes, K., Hynes, R. O. and DeSimone, D. W. (1990) Development 108, 289–298.). Here we report that integrin alpha 3, alpha 4 and alpha 6 mRNA levels increase following induction with 10 U/ml activin-A whereas alpha 5, beta 1 and beta 3 mRNA levels remain unchanged. Whole-mount in situ hybridization reveals that alpha 3 mRNAs are expressed by cells of the involuting mesoderm in the dorsal lip region of early gastrulae. As gastrulation proceeds, alpha 3 expression is localized to a stripe of presumptive notochordal cells along the dorsal midline. In neurulae, alpha 3 mRNA is highly expressed in the notochord but becomes progressively more restricted to the caudalmost portion of this tissue as development proceeds from tailbud to tadpole stages. In addition, alpha 3 is expressed in the forebrain region of later stage embryos. These data suggest that integrin-mediated adhesion may be involved in the process of mesoderm involution at gastrulation and the organization of tissues during embryogenesis.

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Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.5016-5025.1988 ◽

1988 ◽

Vol 8 (11) ◽

pp. 5016-5025

Author(s):

A F Wahl ◽

A M Geis ◽

B H Spain ◽

S W Wong ◽

D Korn ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cell Proliferation ◽

Steady State ◽

Dna Polymerase ◽

Transformed Cells ◽

Mrna Levels ◽

Dna Polymerase Alpha ◽

Human Dna ◽

Alpha Gene

We studied the expression of the human DNA polymerase alpha gene during cell proliferation, during cell progression through the cell cycle, and in transformed cells compared with normal cells. During the activation of quiescent cells (G0 phase) to proliferate (G1/S phases), the steady-state mRNA levels, rate of synthesis of nascent polymerase protein, and enzymatic activity in vitro exhibited a substantial and concordant increase prior to the peak of in vivo DNA synthesis. In transformed cells, the respective values were amplified greater than 10-fold. In actively growing cells separated into discrete stages of the cell cycle by counterflow elutriation or by mitotic shakeoff, levels of steady-state transcripts, translation rates, and enzymatic activities of polymerase alpha were constitutively and concordantly expressed at all stages of the cell cycle, with only a moderate elevation prior to the S phase and a slight decline in the G2 phase. These findings support the conclusion that the regulation of human DNA polymerase alpha gene expression is at the transcriptional level and strongly suggest that the regulatory mechanisms that are operative during the entrance of a cell into the mitotic cycle are fundamentally different from those that modulate polymerase alpha expression in continuously cycling cells.

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Ischemia-reperfusion rapidly increases COX-2 expression in piglet cerebral arteries

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1999.277.3.h1207 ◽

1999 ◽

Vol 277 (3) ◽

pp. H1207-H1214 ◽

Cited By ~ 11

Author(s):

Ferenc Domoki ◽

Roland Veltkamp ◽

Nishadi Thrikawala ◽

Greg Robins ◽

Ferenc Bari ◽

...

Keyword(s):

Ischemia Reperfusion ◽

Cerebral Arteries ◽

Rnase Protection Assay ◽

Immunoblot Analysis ◽

Mrna Levels ◽

Rnase Protection ◽

Time Control ◽

Cox 2 ◽

Cox 1 ◽

Ischemic Stress

In the newborn, cyclooxygenase (COX)-derived products play an important role in the cerebrovascular dysfunction after ischemia-reperfusion (I/R). We examined effects of I/R on expression of COX-1 and COX-2 isoforms in large cerebral arteries of anesthetized piglets. The circle of Willis, the basilar, and the middle cerebral arteries were collected from piglets at 0.5–12 h after global ischemia (2.5–10 min, n = 50), hypoxia ( n = 3), or hypercapnia ( n = 2) and from time-control ( n = 19) or untreated animals ( n = 7). Tissues were analyzed for COX-1 and COX-2 mRNA and protein using RNase protection assay and immunoblot analysis, respectively. Ischemia increased COX-2 mRNA by 30 min, and maximal levels were reached at 2 h. Hypoxia or hypercapnia had minimal effects on COX-2 mRNA. COX-2 protein levels were also consistently elevated by 8 h after I/R. Increases in COX-2 mRNA or protein were not influenced by pretreatment with either indomethacin (5 mg/kg iv, n = 5) or nitro-l-arginine methyl ester (15 mg/kg iv, n = 7). COX-1 mRNA levels were low in time controls, and ischemic stress had no significant effect on COX-1 expression. Thus ischemic stress leads to relatively rapid, selective induction of COX-2 in cerebral arteries.

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Post-transcriptional regulation of bovine parathyroid hormone synthesis

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0100043 ◽

1993 ◽

Vol 10 (1) ◽

pp. 43-49 ◽

Cited By ~ 12

Author(s):

N S Hawa ◽

J L H O'Riordan ◽

S M Farrow

Keyword(s):

Transcriptional Regulation ◽

Steady State ◽

Actinomycin D ◽

Mrna Levels ◽

Dot Blot ◽

Hormone Synthesis ◽

Membrane Bound ◽

Post Transcriptional Regulation ◽

Bovine Parathyroid Hormone ◽

In Cells

ABSTRACT Incubation of bovine parathyroid cells for 48 h in 0·4 mmol calcium/l had no significant effect on steady-state preproparathyroid hormone (preproPTH) mRNA levels when compared with cells incubated in 1·0 mmol calcium/l, but low calcium concentrations increased the membrane-bound polysomal content of preproPTH mRNA by 200±16% (mean±s.d.). No preproPTH mRNA was detected on free polysomes. Actinomycin D (5 and 10 μg/ml) had no effect on steady-state preproPTH mRNA levels measured in dot-blot assays after 24 h, but reduced levels in cells incubated in 1·0 mmol calcium/l to 54±16% and 39±12% of control values respectively after 48 h of incubation. Similarly, in cells incubated in 0·4 mmol calcium/l, actinomycin D (5 and 10μg/ml) reduced steady-state preproPTH mRNA levels to 57±13% and 45±5% of control values respectively. Actinomycin D did not prevent the rise in polysomal content of preproPTH mRNA induced in cells by incubation in 0·4 mmol calcium/l, but increased polysomal content in cells incubated in 0·4 and 1·0mmol calcium/l by 159±9% and 164±13% respectively after 48 h. These results demonstrate post-transcriptional regulation of PTH synthesis in cultured bovine parathyroid cells, and suggest that this control involves a protein which may be calcium-sensitive.

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Interactions between the promoter and first intron are involved in transcriptional control of alpha 1(I) collagen gene expression.

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4851 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4851-4857 ◽

Cited By ~ 68

Author(s):

P Bornstein ◽

J McKay ◽

D J Liska ◽

S Apone ◽

S Devarayalu

Keyword(s):

Growth Hormone ◽

Human Growth Hormone ◽

Transcriptional Control ◽

Rnase Protection Assay ◽

Human Growth ◽

Collagen Gene ◽

Mrna Levels ◽

3T3 Cells ◽

Rnase Protection ◽

First Intron

The first intron of the human collagen alpha 1(I) gene contains several positively and negatively acting elements. We have studied the transcription of collagen-human growth hormone fusion genes, containing deletions and rearrangements of collagen intronic sequences, by transient transfection of chick tendon fibroblasts and NIH 3T3 cells. In chick tendon fibroblasts, but not in 3T3 cells, inversion of intronic sequences containing a previously studied 274-base-pair segment, A274, resulted in markedly reduced human growth hormone mRNA levels as determined by an RNase protection assay. This inhibitory effect was largely alleviated when deletions were introduced in the collagen promoter of plasmids containing negatively oriented intronic sequences. Evidence for interaction of the promoter with the intronic segment, A274, was obtained by gel mobility shift assays. We suggest that promoter-intron interactions, mediated by DNA-binding proteins, regulate collagen gene transcription. Inversion of intronic segments containing critical interactive elements might then lead to an altered geometry and reduced activity of a transcriptional complex in those cells with sufficiently high levels of appropriate transcription factors. We further suggest that the deleted promoter segment plays a key role in directing DNA interactions involved in transcriptional control.

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Regulation of CDC9, the Saccharomyces cerevisiae gene that encodes DNA ligase.

Molecular and Cellular Biology ◽

10.1128/mcb.5.1.226 ◽

1985 ◽

Vol 5 (1) ◽

pp. 226-235 ◽

Cited By ~ 41

Author(s):

T A Peterson ◽

L Prakash ◽

S Prakash ◽

M A Osley ◽

S I Reed

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Steady State ◽

Uv Light ◽

State Level ◽

S Phase ◽

Dna Ligase ◽

Mrna Levels ◽

Steady State Level ◽

High Level

We have cloned CDC9, the structural gene for Saccharomyces cerevisiae DNA ligase, and investigated its transcriptional regulation both as a function of cell cycle stage and after UV irradiation. The steady-state level of DNA ligase mRNA increases at least fourfold in late G1, after the completion of start but before S phase. This high level of CDC9 mRNA then decays with an apparent half-life of ca. 20 min and remains at a low basal level throughout the rest of the cell cycle. The accumulation of CDC9 mRNA in late G1 is dependent upon the completion of start but not the CDC7 and CDC8 functions. Exposure of cells to UV light elicits an eightfold increase in DNA ligase mRNA levels.

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