Complex transcriptional regulation of myc family gene expression in the developing mouse brain and liver

myc family genes (c-, N-, and L-myc) have been shown to be differentially expressed with respect to tissue type and developmental stage. To define and compare the regulatory mechanisms governing their differential developmental expression, we examined the transcriptional regulation of each myc family member during murine postnatal brain and liver development. Nuclear run-on transcription assays demonstrated that both the rate of transcriptional initiation and the degree of transcriptional blocking contribute in a complex manner to the regulation of all three genes. During postnatal brain development, the relative contribution of each transcriptional control mechanism to the regulation of myc family gene expression was found to be different for each gene. For instance, while modulation of transcriptional attenuation did not appear to contribute to the down-regulation of L-myc expression, attenuation was found to be the dominant mechanism by which steady-state N-myc mRNA levels were down-regulated. Different transcriptional strategies were found to be employed in newborn versus adult developing liver for repression of N- and L-myc expression. Undetectable steady-state N- and L-myc mRNA levels in newborn liver were associated with a very low rate of transcriptional initiation, whereas the lack of N- and L-myc expression at the adult stage was accompanied by a high rate of initiation and a striking degree of transcriptional attenuation. Transcriptional attenuation in the N-myc gene was found to map to a region encoding a potential stem-loop structure followed by a thymine tract within the first exon and was not dependent on the use of a specific transcriptional start site.

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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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Regulation of IMP dehydrogenase gene expression by its end products, guanine nucleotides.

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5417 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5417-5425 ◽

Cited By ~ 46

Author(s):

D A Glesne ◽

F R Collart ◽

E Huberman

Keyword(s):

Gene Expression ◽

Steady State ◽

State Level ◽

Mrna Levels ◽

Dependent Manner ◽

Intracellular Level ◽

Nucleotide Analogs ◽

Nucleotide Biosynthesis ◽

Transcriptional Initiation ◽

Imp Dehydrogenase

To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of cellular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time- and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides.

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Regulation of IMP dehydrogenase gene expression by its end products, guanine nucleotides

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5417-5425.1991 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5417-5425

Author(s):

D A Glesne ◽

F R Collart ◽

E Huberman

Keyword(s):

Gene Expression ◽

Steady State ◽

State Level ◽

Mrna Levels ◽

Dependent Manner ◽

Intracellular Level ◽

Nucleotide Analogs ◽

Nucleotide Biosynthesis ◽

Transcriptional Initiation ◽

Imp Dehydrogenase

To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of cellular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time- and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides.

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Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2914-2924.1987 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2914-2924

Author(s):

A Hoekema ◽

R A Kastelein ◽

M Vasser ◽

H A de Boer

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Steady State ◽

Codon Usage ◽

Experimental Approach ◽

Mrna Translation ◽

Yeast Cells ◽

Expression Level ◽

Start Codon ◽

Mrna Levels

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

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Stress mRNA metabolism in canavanine-treated chicken embryo cells

Molecular and Cellular Biology ◽

10.1128/mcb.4.8.1534-1541.1984 ◽

1984 ◽

Vol 4 (8) ◽

pp. 1534-1541

Author(s):

C N White ◽

L E Hightower

Keyword(s):

Steady State ◽

Stress Proteins ◽

Animal Cell ◽

Stress Protein ◽

High Rate ◽

Rrna Synthesis ◽

28S Rrna ◽

Mrna Levels ◽

Molecular Weights

Four major chicken stress mRNAs with apparent molecular weights of 1.2 X 10(6), 0.88 X 10(6), 0.59 X 10(6), and 0.25 X 10(6) to 0.28 X 10(6) were separated on acidic agarose-urea gels. Using cell-free translation, the coding assignments of these mRNAs were determined to be stress proteins with apparent molecular weights of 88,000, 71,000, 35,000, and 23,000. Despite high levels of translational activity in vivo and in vitro, no newly synthesized mRNA for the 23-kilodalton stress protein was detected on gels under conditions which readily allowed detection of other stress mRNAs, suggesting activation of a stored or incompletely processed mRNA. Cloned Drosophila heat shock genes were used to identify and measure changes in cellular levels of the two largest stress mRNAs. Synthesis of these mRNAs increased rapidly during the first hour of canavanine treatment and continued at a high rate for at least 7 h, with the mRNAs attaining new steady-state levels by ca. 3 h. Both of these inducible stress mRNAs had very short half-lives compared with other animal cell mRNAs. Using an approach-to-steady-state analysis, the half-lives were calculated to be 89 min for the mRNA encoding the 88-kilodalton stress protein and 46 min for the mRNA encoding the 71-kilodalton stress protein. Chicken 18S and 28S rRNA synthesis was inhibited, and actin mRNA levels measured with cloned cDNA encoding chicken beta-actin slowly declined in canavanine-treated cells.

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

The Journal of Cell Biology ◽

10.1083/jcb.111.6.2693 ◽

1990 ◽

Vol 111 (6) ◽

pp. 2693-2701 ◽

Cited By ~ 14

Author(s):

J N Feder ◽

C J Guidos ◽

B Kusler ◽

C Carswell ◽

D Lewis ◽

...

Keyword(s):

Cell Cycle ◽

Transcriptional Regulation ◽

Steady State ◽

T Lymphocyte ◽

Ribonucleotide Reductase ◽

Cellular Proliferation ◽

Rnase Protection Assay ◽

Minor Component ◽

Mrna Levels ◽

Rnase Protection

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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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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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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Developmental expression of pIgR gene in sheep mammary gland and hormonal regulation

Journal of Dairy Research ◽

10.1017/s0022029901005362 ◽

2002 ◽

Vol 69 (1) ◽

pp. 13-26 ◽

Cited By ~ 22

Author(s):

AURORE RINCHEV-ALARNOLD ◽

LUCETTE BELAIR ◽

JEAN DJIANE

Keyword(s):

Gene Expression ◽

Mammary Gland ◽

Hormonal Regulation ◽

Mrna Level ◽

Immunohistochemical Analysis ◽

Developmental Expression ◽

Secretory Iga ◽

Mrna Levels ◽

Immunoglobulin Receptor ◽

Pregnancy And Lactation

Secretory IgA found in external secretions are constituted by polymeric IgA (pIgA) bound to the extra-cellular part of the polymeric immunoglobulin receptor (pIgR). The receptor mediates transcytosis of pIgA across epithelial cells. The aim of the present study was to analyse the evolution of pIgR expression in the sheep mammary gland during the development of the mammary gland and to analyse its hormonal regulation. Gene expression of the pIgR was analysed in sheep mammary gland during pregnancy and lactation. By Northern Blot analysis, we observed that low levels of pIgR mRNA are expressed until day 70 of pregnancy. Accumulation of pIgR mRNA started during the third part of pregnancy and intensified 3 d after parturition to reach highest levels during established lactation (day 70). In situ hybridization analysis was used to confirm the increase in pIgR gene expression per mammary epithelial cell. In order to examine the hormonal regulation of the pIgR expression, virgin ewes were hormonally treated. Treatment with oestradiol and progesterone increased pIgR mRNA levels slightly. Subsequent addition of glucocorticoids induced a significant accumulation of pIgR mRNA in the mammary gland of the treated animals. Immunohistochemical analysis was performed to verify that the increase of pIgR mRNA level was associated with enhancement of the pIgR protein in mammary cells. No increase of pIgR mRNA levels were observed if PRL secretion was blocked by bromocryptine injections throughout the hormonal procedure. In conclusion, the present experiments suggest that the enhancement of pIgR levels during lactation result from combined effects of both prolactin and glucocorticoids.

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