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

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.

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

1991 ◽  
Vol 11 (12) ◽  
pp. 6007-6015
Author(s):  
L Xu ◽  
S D Morgenbesser ◽  
R A DePinho
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Steady State ◽  
High Rate ◽  
Developmental Expression ◽  
Tissue Type ◽  
Mrna Levels ◽  
Postnatal Brain ◽  
Transcriptional Initiation ◽  
Family Gene

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.

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

1991 ◽  
Vol 11 (12) ◽  
pp. 6007-6015 ◽  
Author(s):  
L Xu ◽  
S D Morgenbesser ◽  
R A DePinho
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Steady State ◽  
High Rate ◽  
Developmental Expression ◽  
Tissue Type ◽  
Mrna Levels ◽  
Postnatal Brain ◽  
Transcriptional Initiation ◽  
Family Gene

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.

scholarly journals Genomic structure of murine methylmalonyl-CoA mutase: evidence for genetic and epigenetic mechanisms determining enzyme activity

1993 ◽  
Vol 296 (3) ◽  
pp. 663-670 ◽  
Author(s):  
M F Wilkemeyer ◽  
E R Andrews ◽  
F D Ledley
Keyword(s):  
Enzyme Activity ◽  
Steady State ◽  
Transcription Initiation ◽  
Cultured Cells ◽  
Genomic Structure ◽  
Genetic Regulation ◽  
Regulatory Elements ◽  
Transcription Unit ◽  
Mrna Levels ◽  
Sequence Motifs

Methylmalonyl-CoA mutase (MCM) is a nuclear-encoded mitochondrial matrix enzyme. We have reported characterization of murine MCM and cloning of a murine MCM cDNA and now describe the murine Mut locus, its promoter and evidence for tissue-specific variation in MCM mRNA, enzyme and holo-enzyme levels. The Mut locus spans 30 kb and contains 13 exons constituting a unique transcription unit. A B1 repeat element was found in the 3′ untranslated region (exon 13). The transcription initiation site was identified and upstream sequences were shown to direct expression of a reporter gene in cultured cells. The promoter contains sequence motifs characteristic of: (1) TATA-less housekeeping promoters; (2) enhancer elements purportedly involved in co-ordinating expression of nuclear-encoded mitochondrial proteins; and (3) regulatory elements including CCAAT boxes, cyclic AMP-response elements and potential AP-2-binding sites. Northern blots demonstrate a greater than 10-fold variation in steady-state mRNA levels, which correlate with tissue levels of enzyme activity. However, the ratio of holoenzyme to total enzyme varies among different tissues, and there is no correlation between steady-state mRNA levels and holoenzyme activity. These results suggest that, although there may be regulation of MCM activity at the level of mRNA, the significance of genetic regulation is unclear owning to the presence of epigenetic regulation of holoenzyme formation.

scholarly journals Gene Expression of Nucleic Acid-Sensing Pattern Recognition Receptors in Children Hospitalized for Respiratory Syncytial Virus-Associated Acute Bronchiolitis

2009 ◽  
Vol 16 (6) ◽  
pp. 816-823 ◽  
Author(s):  
Carolina Scagnolari ◽  
Fabio Midulla ◽  
Alessandra Pierangeli ◽  
Corrado Moretti ◽  
Enea Bonci ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pattern Recognition ◽  
Viral Load ◽  
Respiratory Syncytial Virus ◽  
Pattern Recognition Receptors ◽  
Mrna Levels ◽  
Acute Bronchiolitis ◽  
Expression Levels ◽  
Rsv Infection ◽  
Syncytial Virus

ABSTRACT Given the critical role of pattern recognition receptors (PRRs) in acid nucleic recognition in the initiation of innate immunity and the orchestration of adaptive immunity, the aim of this study was to determine whether any heterogeneity of PRR expression in the airway tracts of infants with respiratory syncytial virus (RSV) infection might explain the broad clinical spectrum of RSV-associated bronchiolitis in infants. For this purpose, the levels of melanoma differentiation-associated protein-5 (MDA-5), retinoic acid inducible gene-1 (RIG-1), and Toll-like receptor 3 (TLR-3), TLR-7, TLR-8, and TLR-9 mRNAs were evaluated, using TaqMan quantitative reverse transcription-PCR, in cells from nasopharyngeal washes collected from 157 infants suffering from acute bronchiolitis whether or not they were associated with respiratory viruses. High interindividual variability was observed in both virus-positive and -negative infants; however, the relative gene expression levels of MDA-5, RIG-1, TLR-7, and TLR-8 were significantly higher in the virus-infected group, whereas the expression levels of TLR-3 and TLR-9 were not significantly different. The differences in the gene expression of MDA-5, RIG-1, TLR-7, and TLR-8 were more evident in infants with RSV infection than in those with bocavirus or rhinovirus infection. In RSV-infected infants, PRR-mRNA levels also were analyzed in relation to interferon protein levels, viral load, clinical severity, days of hospitalization, age, and body weight. A significant positive correlation was observed only between RSV viral load and RIG-1 mRNA levels. These findings provide the first direct evidence that, in infants with respiratory virus-associated bronchiolitis, especially RSV, there are substantial changes in PRR gene expression; this likely is an important determinant of the clinical outcome of bronchiolitis.

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

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.

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

1990 ◽  
Vol 111 (6) ◽  
pp. 2693-2701 ◽  
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.

scholarly journals Regulation of asparagine synthetase gene expression by amino acid starvation

1991 ◽  
Vol 11 (12) ◽  
pp. 6059-6066
Author(s):  
S S Gong ◽  
L Guerrini ◽  
C Basilico
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Transcription Initiation ◽  
Rna Stability ◽  
Genomic Region ◽  
Amino Acid Starvation ◽  
Asparagine Synthetase ◽  
Temperature Sensitive ◽  
Mrna Levels ◽  
Regulation Of Expression

We have studied the regulation of expression of the asparagine synthetase (AS) gene in ts11 cells, a mutant of BHK hamster cells which encodes a temperature-sensitive AS and therefore does not produce endogenous asparagine at 39.5 degrees C. Incubation of ts11 cells at the nonpermissive temperature drastically increases the level of AS mRNA, and the stimulation of AS mRNA expression is effectively suppressed by the addition of asparagine to the medium. We show here that regulation of AS gene expression involves cis-acting elements which are contained in the mRNA as well as in the 5' genomic region. When a plasmid containing the human AS cDNA under the control of the human AS promoter region was stably transfected into ts11 cells, the expression of human AS RNAs was regulated as that of the endogenous hamster transcripts, indicating that this construct contained all cis elements necessary for regulation. Expression of the AS cDNA in ts11 cells under the control of a constitutive foreign promoter was also regulated by the concentration of asparagine, and this regulation required translation. When we introduced by mutagenesis a number of stop codons in the AS cDNA, the mutant mRNAs with short open reading frames were expressed at low levels that were not increased by asparagine deprivation. Inhibition of protein and RNA synthesis also prevented down-regulation of AS mRNA levels by high concentrations of asparagine. In a parallel series of experiments, we showed that an AS DNA fragment including the promoter and first exon can also regulate RNA expression in response to asparagine concentration. Furthermore, similar increases in the levels of AS RNAs are produced not only by asparagine deprivation in ts11 cells but also by deprivation of human and wild-type BHK cells of leucine, isoleucine, or glutamine. Thus, regulation of AS gene expression is a response to amino acid starvation through mechanisms which appear to involve both changes in RNA stability and change in the rates of transcription initiation or elongation.

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

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.

Post-transcriptional regulation of bovine parathyroid hormone synthesis

1993 ◽  
Vol 10 (1) ◽  
pp. 43-49 ◽  
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.

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

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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