scholarly journals An 11-nt sequence polymorphism at the 3′UTR of human SFTPA1 and SFTPA2 gene variants differentially affect gene expression levels and miRNA regulation in cell culture

2014 ◽  
Vol 307 (1) ◽  
pp. L106-L119 ◽  
Author(s):  
Patricia Silveyra ◽  
Susan L. DiAngelo ◽  
Joanna Floros
Keyword(s):  
Gene Expression ◽  
Chinese Hamster Ovary Cells ◽  
Regulation Of Gene Expression ◽  
Chinese Hamster ◽  
Normal Lung ◽  
Gene Variants ◽  
Translation Efficiency ◽  
Mrna Levels ◽  
Mirna Regulation

Surfactant protein A (SP-A) plays a vital role in maintaining normal lung function and in host defense. Two genes encode SP-A in humans (SFTPA1, SFTPA2), and several gene variants have been identified for these. We have previously shown that sequence elements of SFTPA1 and SFTPA2 3′ untranslated regions (UTRs) differentially affect translation efficiency in vitro. Polymorphisms at the 3′UTRs of mRNA variants may account for differential binding of miRNAs, a class of small noncoding RNAs that regulate gene expression. In this work, we generated 3′UTR reporter constructs of the SFTPA1 and SFTPA2 variants most frequently found in the population, as well as mutants of a previously described 11-nt indel element (refSNP rs368700152). Reporter constructs were transfected in NCI-H441 cells in the presence or absence of miRNA mimics, and reporter gene expression was analyzed. We found that human miRNA mir-767 negatively affected expression of constructs containing SFTPA1 and SFTPA2 variants, whereas mir-4507 affected only constructs with 3′UTRs of SFTPA1 variants 6A, 6A3, and 6A4 (not containing the 11-nt element). Three miRNAs (mir-183, mir-449b, and mir-612) inhibited expression of recombinants of SFTPA2 variants and the SFTPA1 variant 6A2, all containing the 11-nt element. Similar results were obtained for SP-A expression when these miRNAs were transfected in Chinese hamster ovary cells expressing SFTPA1 or SFTPA2 variants or in NCI-H441 cells (genotype 1A5/1A5-6A4/6A4). Moreover, transfection with a specific antagomir (antagomir-183) reversed the effects of mir-183 on SP-A mRNA levels. Our results indicate that sequence variability at the 3′UTR of SP-A variants differentially affects miRNA regulation of gene expression.

scholarly journals Effects of Altering the Transcription Termination Signals of Respiratory Syncytial Virus on Viral Gene Expression and Growth In Vitro and In Vivo

2004 ◽  
Vol 78 (2) ◽  
pp. 692-699 ◽  
Author(s):  
Kim C. Tran ◽  
Peter L. Collins ◽  
Michael N. Teng
Keyword(s):  
Gene Expression ◽  
Respiratory Syncytial Virus ◽  
Respiratory Tract ◽  
Transcription Termination ◽  
Regulation Of Gene Expression ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Mrna Levels ◽  
Syncytial Virus

ABSTRACT Nonsegmented negative-sense RNA viruses (mononegaviruses) control viral gene expression largely through a transcription gradient such that promoter-proximal genes are transcribed more abundantly than downstream genes. For some paramyxoviruses, naturally occurring differences in the levels of efficiency of transcription termination by various gene end (GE) signals provide an additional level of regulation of gene expression. The first two genes (NS1 and NS2) of respiratory syncytial virus (RSV) are particularly inefficient in termination. We investigated whether altering the termination efficiency (TE) of these two genes in infectious recombinant virus would affect transcription of promoter-proximal and promoter-distal genes, production of viral proteins, and viral replication in cell culture and in the respiratory tract of mice. Recombinant RSVs were constructed with mutations that increased or decreased the TE of the NS1 GE signal, increased that of the NS2 GE signal, or increased that of both signals. Increasing the TE of either or both GE signals resulted in decreased production of the related polycistronic readthrough mRNAs, which normally arise due to the failure of the viral polymerase to recognize the GE signal. This was accompanied by a small increase in the levels of monocistronic NS1 and NS2 mRNAs. Conversely, decreasing the TE of the NS1 GE increased the production of readthrough mRNAs concomitant with a decrease of monocistronic NS1 and NS2 mRNA levels. These changes were reflected in the levels of NS1 and NS2 protein. All of the mutant viruses displayed growth kinetics and virus yields similar to wild-type recombinant RSV (rA2) in both HEp-2 and Vero cells. In addition, all mutants grew similarly to rA2 in the upper- and lower-respiratory tract of BALB/c mice, though some of the mutants displayed slightly decreased replication. These data suggest that the natural inefficiencies of transcription termination by the NS1 and NS2 GE signals do not play important roles in controlling the magnitude of RSV gene expression or the efficiency of virus replication. Furthermore, while changes in the TE of a GE signal clearly can affect the transcription of its gene as well as that of the one immediately downstream, these changes did not have a significant effect on the overall transcriptional gradient.

scholarly journals The Ferredoxin-Like Protein FerR Regulates PrbP Activity inLiberibacter asiaticus

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Lei Pan ◽  
Danilo da Silva ◽  
Fernando A. Pagliai ◽  
Natalie A. Harrison ◽  
Claudio F. Gonzalez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Regulation Of Gene Expression ◽  
Accessory Protein ◽  
Mrna Levels ◽  
Plant Host ◽  
Content Type ◽  
Environmental Signals ◽  
Liberibacter Asiaticus

ABSTRACTInLiberibacter asiaticus, PrbP is an important transcriptional accessory protein that regulates gene expression through interactions with the RNA polymerase β-subunit and a specific sequence on the promoter region. The constitutive expression ofprbPobserved upon chemical inactivation of PrbP-DNA interactionsin vivoindicated that the expression ofprbPwas not autoregulated at the level of transcription. This observation suggested that a modulatory mechanism via protein-protein interactions may be involved.In silicogenome association analysis identified FerR (CLIBASIA_01505), a putative ferredoxin-like protein, as a PrbP-interacting protein. Using a bacterial two-hybrid system and immunoprecipitation assays, interactions between PrbP and FerR were confirmed.In vitrotranscription assays were used to show that FerR can increase the activity of PrbP by 16-fold when present in the PrbP-RNA polymerase reaction mixture. The FerR protein-protein interaction surface was predicted by structural modeling and followed by site-directed mutagenesis. Amino acids V20, V23, and C40 were identified as the most important residues in FerR involved in the modulation of PrbP activityin vitro. The regulatory mechanism of FerR abundance was examined at the transcription level. In contrast toprbPofL. asiaticus(prbPLas), mRNA levels offerRofL. asiaticus(ferRLas) are induced by an increase in osmotic pressure. The results of this study revealed that the activity of the transcriptional activator PrbPLasis modulated via interactions with FerRLas. The induction offerRLasexpression by osmolarity provides insight into the mechanisms of adjusting gene expression in response to host environmental signals inL. asiaticus.IMPORTANCEThe rapid spread and aggressive progression of huanglongbing (HLB) in the major citrus-producing areas have raised global recognition of and vigilance to this disease. As a result, the causative agent,Liberibacter asiaticus, has been investigated from various perspectives. However, gene expression regulatory mechanisms that are important for the survival and persistence of this intracellular pathogen remain largely unexplored. PrbP is a transcriptional accessory protein important forL. asiaticussurvival in the plant host. In this study, we investigated the interactions between PrbP inL. asiaticus(PrbPLas) and a ferredoxin-like protein (FerR) inL. asiaticus, FerRLas. We show that the presence of FerR stabilizes and augments the activity of PrbPLas. In addition, we demonstrate that the expression offerRis induced by increases in osmolarity inLiberibacter crescens. Altogether, these results suggest that FerRLasand PrbPLasmay play important roles in the regulation of gene expression in response to changing environmental signals duringL. asiaticusinfection in the citrus host.

scholarly journals Secondary Structure of Chloroplast mRNAs In Vivo and In Vitro

Plants ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 323
Author(s):  
Piotr Gawroński ◽  
Aleksandra Pałac ◽  
Lars B. Scharff
Keyword(s):  
Gene Expression ◽  
Secondary Structure ◽  
Translation Initiation ◽  
Structural Information ◽  
Regulation Of Gene Expression ◽  
Dimethyl Sulfate ◽  
Translation Efficiency ◽  
Mrna Secondary Structure

mRNA secondary structure can influence gene expression, e.g., by influencing translation initiation. The probing of in vivo mRNA secondary structures is therefore necessary to understand what determines the efficiency and regulation of gene expression. Here, in vivo mRNA secondary structure was analyzed using dimethyl sulfate (DMS)-MaPseq and compared to in vitro-folded RNA. We used an approach to analyze specific, full-length transcripts. To test this approach, we chose low, medium, and high abundant mRNAs. We included both monocistronic and multicistronic transcripts. Because of the slightly alkaline pH of the chloroplast stroma, we could probe all four nucleotides with DMS. The structural information gained was evaluated using the known structure of the plastid 16S rRNA. This demonstrated that the results obtained for adenosines and cytidines were more reliable than for guanosines and uridines. The majority of mRNAs analyzed were less structured in vivo than in vitro. The in vivo secondary structure of the translation initiation region of most tested genes appears to be optimized for high translation efficiency.

scholarly journals The untranslated exon B of human surfactant protein A2 mRNAs is an enhancer for transcription and translation

2011 ◽  
Vol 301 (5) ◽  
pp. L795-L803 ◽  
Author(s):  
Patricia Silveyra ◽  
Manmeet Raval ◽  
Brett Simmons ◽  
Susan DiAngelo ◽  
Guirong Wang ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Splice Variants ◽  
Random Sequence ◽  
Regulation Of Gene Expression ◽  
Surfactant Protein ◽  
Regulatory Elements ◽  
Translation Efficiency ◽  
Mrna Levels ◽  
Position And Orientation

Two human genes, SFTPA1 (SP-A1) and SFTPA2 (SP-A2), encode surfactant protein A, a molecule of innate immunity and surfactant-related functions. Several genetic variants have been identified for both genes. These include nucleotide (nt) polymorphisms, as well as alternative splicing patterns at the 5′ untranslated region (5′UTR). Exon B (eB) is included in the 5′UTR of most SP-A2, but not SP-A1 splice variants. We investigated the role of eB in the regulation of gene expression and translation efficiency. A luciferase (Luc) reporter gene was cloned downstream of the entire (AeBD) or eB deletion mutants (del_mut) of the SP-A2 5′UTR, or heterologous 5′UTRs containing the eB sequence, or a random sequence of equal length. The del_mut constructs consisted in consecutive deletions of five nucleotides ( n = 8) within eB and the exon-exon junctions in the AeBD 5′UTR. Luc activities and mRNA levels were compared after transfection of NCI-H441 cells. We found that 1) eB increased Luc mRNA levels when placed upstream of heterologous 5′UTR sequences or the promoter region, regardless of its position and orientation; 2) translation efficiency of in vitro-generated mRNAs containing eB was higher than that of mRNAs without eB; and 3) the integrity of eB sequence is crucial for transcription and translation of the reporter gene. Thus eB 1) is a transcription enhancer, because it increases mRNA content regardless of position and orientation, 2) enhances translation when placed in either orientation within its natural 5′UTR sequence and in heterologous 5′UTRs, and 3) contains potential regulatory elements for both transcription and translation. We conclude that eB sequence and length are determinants of transcription and translation efficiency.

scholarly journals G-quadruplex RNA motifs influence gene expression in the malaria parasite Plasmodium falciparum

2021 ◽  
Author(s):  
Franck Dumetz ◽  
Eugene Yui-Ching Chow ◽  
Lynne M. Harris ◽  
Mubarak I. Umar ◽  
Anders Jensen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Plasmodium Falciparum ◽  
Malaria Parasite ◽  
Regulation Of Gene Expression ◽  
Low Complexity ◽  
Telomere Maintenance ◽  
Translation Efficiency ◽  
G Quadruplex

ABSTRACTG-quadruplexes are non-helical secondary structures that can fold in vivo in both DNA and RNA. In human cells, they can influence replication, transcription and telomere maintenance in DNA, or translation, transcript processing and stability of RNA. We have previously showed that G-quadruplexes are detectable in the DNA of the malaria parasite Plasmodium falciparum, despite a very highly A/T-biased genome with unusually few guanine-rich sequences. Here, we show that RNA G-quadruplexes can also form in P. falciparum RNA, using rG4-seq for transcriptome-wide structure-specific RNA probing. Many of the motifs, detected here via the rG4seeker pipeline, have non-canonical forms and would not be predicted by standard in silico algorithms. However, in vitro biophysical assays verified the formation of non-canonical motifs. The G-quadruplexes in the P. falciparum transcriptome are frequently clustered in certain genes and associated with regions encoding low-complexity peptide repeats. They are overrepresented in particular classes of genes, notably those that encode PfEMP1 virulence factors, stress response genes and DNA binding proteins. In vitro translation experiments and in vivo measures of translation efficiency showed that G-quadruplexes can influence the translation of P. falciparum mRNAs. Thus, the G-quadruplex is a novel player in post-transcriptional regulation of gene expression in this major human pathogen.

Design, Synthesis, Molecular Docking and Biological Evaluation of 1-(benzo[d]thiazol-2-ylamino)(phenyl)methyl)naphthalen-2-ol Derivatives as Antiproliferative Agents

2019 ◽  
Vol 16 (10) ◽  
pp. 837-845
Author(s):  
Sandhya Jonnala ◽  
Bhaskar Nameta ◽  
Murthy Chavali ◽  
Rajashaker Bantu ◽  
Pallavi Choudante ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Inhibitory Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mouse Skin ◽  
Coupling Reaction ◽  
Binding Mode ◽  
Biological Evaluation ◽  
Design Synthesis

A class of 1-((benzo[d]thiazol-2-ylamino)(phenyl)methyl)naphthalen-2-ol derivatives (4a-t) has been synthesized in good yields through a three component coupling reaction. The newly synthesized compounds were evaluated for their in vitro antiproliferative activity against five cell lines such as DU145 (human prostate cancer), MDA-MB-B231 (human breast cancer), SKOV3 (human ovarian cancer), B16-F10 (mouse skin melanoma) and CHO-K1 (Chinese hamster ovary cells), a noncancerous cell line. In vitro inhibitory activity indicates that compounds 4a, 4b, 4c, 4d, 4g, 4j, and 4o exhibited potent anti-proliferative behavior. Among them, compounds 4g, 4j and 4o found to be the most active members exhibiting remarkable growth inhibitory activity. Molecular docking facilitates to investigate the probable binding mode and key active site interactions in tubulins α and β proteins. The docking results are complementary to experimental results.

scholarly journals In Vitro Cytotoxic Activity Guided Essential Oil Composition of Flowering Twigs of Stevia rebaudiana

2014 ◽  
Vol 9 (5) ◽  
pp. 1934578X1400900 ◽  
Author(s):  
Tavleen S Mann ◽  
Vijai K Agnihotri ◽  
Dharmesh Kumar ◽  
Probir K Pal ◽  
Rajkesh Koundal ◽  
...  
Keyword(s):  
Essential Oil ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complex Mixture ◽  
Stevia Rebaudiana ◽  
Spectroscopic Techniques ◽  
Oil Composition ◽  
Standard Drug ◽  
Rat Glioma

The essential oil extracted by hydrodistillation from the flowering twigs of Stevia rebaudiana Bertoni (Asteraceae) was fractioned by chromatography. Forty-three constituents were characterized with the help of GC, GC-MS and other spectroscopic techniques. The essential oil was found to be a complex mixture of mono- and sesqui-terpenes. The cytotoxicity of the essential oil and its fractions was evaluated by sulforhodamine B (SRB) based assay against two cancer cell types viz. C-6 (rat glioma cells) and CHOK1 (Chinese hamster ovary cells). The essential oil and its fractions showed promising cytotoxicity against both cell lines. The highest activity (95.6±0.6%) was show by the essential oil on the C-6 cell line at a concentration of 400 μg/mL, which was comparable with that of the standard drug vinblastin.

scholarly journals Evaluation of Fab and F(ab′)2 Fragments and Isotype Variants of a Recombinant Human Monoclonal Antibody against Shiga Toxin 2

2010 ◽  
Vol 78 (3) ◽  
pp. 1376-1382 ◽  
Author(s):  
Donna E. Akiyoshi ◽  
Abhineet S. Sheoran ◽  
Curtis M. Rich ◽  
L. Richard ◽  
Susan Chapman-Bonofiglio ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Shiga Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Human Monoclonal Antibody ◽  
Chinese Hamster ◽  
The Body ◽  
Neutralization Activity ◽  
Shiga Toxin 2

ABSTRACT 5C12 HuMAb is a human monoclonal antibody against the A subunit of Shiga toxin 2 (Stx2). We have previously shown that 5C12 HuMAb effectively neutralizes the cytotoxic effects of this toxin by redirecting its transport within the cell and also by neutralizing the toxin's ability to inhibit protein synthesis. The 5C12 HuMAb and its recombinant IgG1 version protect mice at a dose of 0.6 μg against a lethal challenge of Stx2. The contribution of the Fc region to this observed neutralization activity of the 5C12 antibody against Stx2 was investigated in this study. Using recombinant DNA technology, 5C12 isotype variants (IgG1, IgG2, IgG3, and IgG4) and antibody fragments [Fab, F(ab′)2] were expressed in Chinese hamster ovary cells and evaluated in vitro and in vivo. All four 5C12 isotype variants showed protection in vitro, with the IgG3 and IgG4 variants showing the highest protection in vivo. The Fab and F(ab′)2 fragments also showed protection in vitro but no protection in the mouse toxicity model. Similar results were obtained for a second HuMAb (5H8) against the B subunit of Stx2. The data suggest the importance of the Fc region for neutralization activity, but it is not clear if this is related to the stability of the full-length antibody or if the Fc region is required for effective elimination of the toxin from the body.

scholarly journals Differential Effects of Gonadotropin-Releasing Hormone (GnRH) Pulse Frequency on Gonadotropin Subunit and GnRH Receptor Messenger Ribonucleic Acid Levels in Vitro*

Endocrinology ◽  
1997 ◽  
Vol 138 (3) ◽  
pp. 1224-1231 ◽  
Author(s):  
Ursula B. Kaiser ◽  
Andrzej Jakubowiak ◽  
Anna Steinberger ◽  
William W. Chin
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Pulse Frequency ◽  
Gnrh Receptor ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Subunit Gene ◽  
Differential Effects ◽  
Messenger Ribonucleic Acid

Abstract The hypothalamic hormone, GnRH, is released and transported to the anterior pituitary in a pulsatile manner, where it binds to specific high-affinity receptors and regulates gonadotropin biosynthesis and secretion. The frequency of GnRH pulses changes under various physiological conditions, and varying GnRH pulse frequencies have been shown to regulate differentially the secretion of LH and FSH and the expression of the gonadotropin α, LHβ, and FSHβ subunit genes in vivo. We demonstrate differential effects of varying GnRH pulse frequency in vitro in superfused primary monolayer cultures of rat pituitary cells. Cells were treated with 10 nm GnRH pulses for 24 h at a frequency of every 0.5, 1, 2, or 4 h. α, LHβ, and FSHβ messenger RNA (mRNA) levels were increased by GnRH at all pulse frequencies. α and LHβ mRNA levels and LH secretion were stimulated to the greatest extent at a GnRH pulse frequency of every 30 min, whereas FSHβ mRNA levels and FSH secretion were stimulated maximally at a lower GnRH pulse frequency, every 2 h. GnRH receptor (GnRHR) mRNA levels also were increased by GnRH at all pulse frequencies and were stimulated maximally at a GnRH pulse frequency of every 30 min. Similar results were obtained when the dose of each pulse of GnRH was adjusted to maintain a constant total cumulative dose of GnRH over 24 h. These data show that gonadotropin subunit gene expression is regulated differentially by varying GnRH pulse frequencies in vitro, suggesting that the differential effects of varying GnRH pulse frequencies on gonadotropin subunit gene expression occur directly at the level of the pituitary. The pattern of regulation of GnRHR mRNA levels correlated with that of α and LHβ but was different from that of FSHβ. This suggests that α and LHβ mRNA levels are maximally stimulated when GnRHR levels are relatively high, whereas FSHβ mRNA levels are maximally stimulated at lower levels of GnRHR expression, and that the mechanism for differential regulation of the gonadotropins by varying pulse frequencies of GnRH may involve levels of GnRHR. Furthermore, these data suggest that the mechanisms whereby varying GnRH pulse frequencies stimulate α, LHβ, and GnRHR gene expression are similar, whereas the stimulation of FSHβ mRNA levels may be different.

Sign in / Sign up

Export Citation Format

Share Document