-
Gene Jan 2022Abnormal expression of ionotropic glutamate receptor NMDA type subunit 1, the key subunit of the NMDA receptor, may be related to many neuropsychiatric disorders. In...
INTRODUCTION
Abnormal expression of ionotropic glutamate receptor NMDA type subunit 1, the key subunit of the NMDA receptor, may be related to many neuropsychiatric disorders. In this study, we explored the functional sequence of the 5' regulatory region of the human GRIN1 gene and discussed the transcription factors that may regulate gene expression.
MATERIALS AND METHODS
Twelve recombinant pGL3 vectors with gradually truncated fragment lengths were constructed, transfected into HEK-293, U87, and SK-N-SH cell lines, and analyzed through the luciferase reporter gene assay. JASPAR database is used to predict transcription factors.
RESULTS
In SK-N-SH and U87 cell lines, regions from -337 to -159 bp, -704 to -556 bp inhibited gene expression, while -556 to -337 bp upregulated gene expression. In HEK-293 and U87 cell lines, the expression of fragment -1703 to + 188 bp was significantly increased compared to adjacent fragments -1539 to + 188 bp and -1843 to + 188 bp. The protein expressions of fragments -2162 to + 188 bp and -2025 to + 188 bp, -1539 to + 188 bp and -1215 to + 188 bp, -1215 to + 188 bp and -1066 to + 188 bp were significantly different in HEK-293 and SK-N-SH cells. According to the predictions of the JASPAR database, the transcription factors REST, EGR1, and CREB1/HIC2 may bind the DNA sequences of GRIN1 gene from the -337 to -159, -556 to -337, and -704 to -556, respectively. In addition, zinc finger transcription factors may regulate the expression of other differentially expressed fragments.
CONCLUSIONS
Abnormal transcription regulation in the proximal promoter region of GRIN1 (-704 to + 188 bp) may be involved in the course of neuropsychiatric diseases.
Topics: 5' Untranslated Regions; Cell Line, Tumor; Gene Expression; Gene Expression Regulation; Genes, Reporter; HEK293 Cells; Humans; Nerve Tissue Proteins; Promoter Regions, Genetic; Receptors, N-Methyl-D-Aspartate; Transcription Factors; Transcription, Genetic; Transcriptional Activation
PubMed: 34592350
DOI: 10.1016/j.gene.2021.145973 -
Journal of Virology Feb 2019Engineered recombinant viruses expressing reporter genes have been developed for real-time monitoring of replication and for mass screening of antiviral inhibitors....
Engineered recombinant viruses expressing reporter genes have been developed for real-time monitoring of replication and for mass screening of antiviral inhibitors. Recently, we reported using a reverse genetics system to develop the first recombinant reporter rotaviruses (RVs) that expressed NanoLuc (NLuc) luciferase. Here, we describe a strategy for developing stable reporter RVs expressing luciferase and green or red fluorescent proteins. The reporter genes were inserted into the open reading frame of NSP1 and expressed as a fusion with an NSP1 peptide consisting of amino acids 1 to 27. The stability of foreign genes within the reporter RV strains harboring a shorter chimeric NSP1-reporter gene was greater than that of those in the original reporter RV strain, independent of the transgene inserted. The improved reporter RV was used to screen for neutralizing monoclonal antibodies (MAbs). Sequence analysis of escape mutants from one MAb clone (clone 29) identified an amino acid substitution (arginine to glycine) at position 441 in the VP4 protein, which resides within neutralizing epitope 5-1 in the VP5* fragment. Furthermore, to express a native reporter protein lacking NSP1 amino acids 1 to 27, the 5'- and 3'-terminal region sequences were modified to restore the predicted secondary RNA structure of the NSP1-reporter chimeric gene. These data demonstrate the utility of reporter RVs for live monitoring of RV infections and also suggest further applications (e.g., RV vaccine vectors, which can induce mucosal immunity against intestinal pathogens). Development of reporter RVs has been hampered by the lack of comprehensive reverse genetics systems. Recently, we developed a plasmid-based reverse genetics system that enables generation of reporter RVs expressing NLuc luciferase. The prototype reporter RV had some disadvantages (i.e., the transgene was unstable and was expressed as a fusion protein with a partial NSP1 peptide); however, the improved reporter RV overcomes these problems through modification of the untranslated region of the reporter-NSP1 chimeric gene. This strategy for generating stable reporter RVs could be expanded to diverse transgenes and be used to develop RV transduction vectors. Also, the data improve our understanding of the importance of 5'- and 3'-terminal sequences in terms of genome replication, assembly, and packaging.
Topics: Cell Line; Gene Expression; Gene Transfer Techniques; Genes, Reporter; Luciferases; Plasmids; Rotavirus; Rotavirus Infections; Virus Replication
PubMed: 30541830
DOI: 10.1128/JVI.01774-18 -
European Review For Medical and... Sep 2021In our previous genomic studies in human intracranial aneurysms, we observed downregulations in the expression of a number of ribosomal protein genes and the...
OBJECTIVE
In our previous genomic studies in human intracranial aneurysms, we observed downregulations in the expression of a number of ribosomal protein genes and the c-Myc-related gene MYC target 1 (MYCT1). So far there is no information about the roles of MYCT1 in vascular cells. Our study aims to investigate the functional roles of MYCT1 in vascular smooth muscle cells (SMCs).
MATERIALS AND METHODS
Primary SMCs were isolated from rat thoracic aorta and cultured in vitro. The mRNA and protein expressions were determined by real-time PCR and western blot respectively. Apoptosis was detected by measuring caspase 3/7 activity. Collagen production was determined with ELISA.
RESULTS
Using PCR, we validated our previous genomic data showing that the expressions of MYCT1 and ribosomal protein genes were decreased in human aneurysm tissues. In vascular SMCs, we showed that nitrosative stress downregulated the expression of both MYCT1 and ribosomal proteins. Knockdown of MYCT1 mimicked the effects of nitrosative stress on ribosomal protein expressions, whereas overexpression of MYCT1 blunted the effects of nitrosative stress. MYCT1-dependent downregulation of ribosomal proteins compromised the protein translational capacity of the cells for collagen production. Moreover, the endogenously expressed MYCT1 in vascular SMCs was involved in maintaining normal cellular functions including survival, proliferation and migration.
CONCLUSIONS
MYCT1-dependent gene regulation may, at least partly, explain the downregulated expressions of ribosomal proteins observed in human intracranial aneurysms. It is suggested that MYCT1 may represent a novel molecular target for counteracting the decreased activity of aneurysmal SMCs for tissue repairmen/regeneration.
Topics: Animals; Cells, Cultured; Down-Regulation; Gene Expression; Gene Expression Regulation; Humans; Intracranial Aneurysm; Male; Muscle, Smooth, Vascular; Nerve Regeneration; Nitrosative Stress; Nuclear Proteins; Rats, Sprague-Dawley; Ribosomal Proteins; Rats
PubMed: 34604957
DOI: 10.26355/eurrev_202109_26784 -
Scientific Reports Jun 2015Differential mRNA expression studies implicitly assume that changes in mRNA expression have biological meaning, most likely mediated by corresponding changes in protein...
Differential mRNA expression studies implicitly assume that changes in mRNA expression have biological meaning, most likely mediated by corresponding changes in protein levels. Yet studies into mRNA-protein correspondence have shown notoriously poor correlation between mRNA and protein expression levels, creating concern for inferences from only mRNA expression data. However, none of these studies have examined in particular differentially expressed mRNA. Here, we examined this question in an ovarian cancer xenograft model. We measured protein and mRNA expression for twenty-nine genes in four drug-treatment conditions and in untreated controls. We identified mRNAs differentially expressed between drug-treated xenografts and controls, then analysed mRNA-protein expression correlation across a five-point time-course within each of the four experimental conditions. We evaluated correlations between mRNAs and their protein products for mRNAs differentially expressed within an experimental condition compared to those that are not. We found that differentially expressed mRNAs correlate significantly better with their protein product than non-differentially expressed mRNAs. This result increases confidence for the use of differential mRNA expression for biological discovery in this system, as well as providing optimism for the usefulness of inferences from mRNA expression in general.
Topics: Animals; Female; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Heterografts; Mice; Mice, Nude; Ovarian Neoplasms; Proteins; RNA, Messenger
PubMed: 26053859
DOI: 10.1038/srep10775 -
Brain Research May 2016NG2(+) glial cells are a dynamic population of non-neuronal cells that give rise to myelinating oligodendrocytes in the central nervous system. These cells express... (Review)
Review
NG2(+) glial cells are a dynamic population of non-neuronal cells that give rise to myelinating oligodendrocytes in the central nervous system. These cells express numerous ion channels and neurotransmitter receptors, which endow them with a complex electrophysiological profile that is unique among glial cells. Despite extensive analysis of the electrophysiological properties of these cells, relatively little was known about the molecular identity of the channels and receptors that they express. The generation of new RNA-Seq datasets for NG2(+) cells has provided the means to explore how distinct genes contribute to the physiological properties of these progenitors. In this review, we systematically compare the results obtained through RNA-Seq transcriptional analysis of purified NG2(+) cells to previous physiological and molecular studies of these cells to define the complement of ion channels and neurotransmitter receptors expressed by NG2(+) cells in the mammalian brain and discuss the potential significance of the unique physiological properties of these cells. This article is part of a Special Issue entitled SI:NG2-glia(Invited only).
Topics: Animals; Antigens; Gene Expression; Humans; Neuroglia; Proteoglycans; Receptors, Neurotransmitter
PubMed: 26385417
DOI: 10.1016/j.brainres.2015.09.010 -
BMC Genomics Oct 2015A complete understanding of barnacle adhesion remains elusive as the process occurs within and beneath the confines of a rigid calcified shell. Barnacle cement is mainly...
BACKGROUND
A complete understanding of barnacle adhesion remains elusive as the process occurs within and beneath the confines of a rigid calcified shell. Barnacle cement is mainly proteinaceous and several individual proteins have been identified in the hardened cement at the barnacle-substrate interface. Little is known about the molt- and tissue-specific expression of cement protein genes but could offer valuable insight into the complex multi-step processes of barnacle growth and adhesion.
METHODS
The main body and sub-mantle tissue of the barnacle Amphibalanus amphitrite (basionym Balanus amphitrite) were collected in pre- and post-molt stages. RNA-seq technology was used to analyze the transcriptome for differential gene expression at these two stages and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) was used to analyze the protein content of barnacle secretions.
RESULTS
We report on the transcriptomic analysis of barnacle cement gland tissue in pre- and post-molt growth stages and proteomic investigation of barnacle secretions. While no significant difference was found in the expression of cement proteins genes at pre- and post-molting stages, expression levels were highly elevated in the sub-mantle tissue (where the cement glands are located) compared to the main barnacle body. We report the discovery of a novel 114kD cement protein, which is identified in material secreted onto various surfaces by adult barnacles and with the encoding gene highly expressed in the sub-mantle tissue. Further differential gene expression analysis of the sub-mantle tissue samples reveals a limited number of genes highly expressed in pre-molt samples with a range of functions including cuticular development, biominerialization, and proteolytic activity.
CONCLUSIONS
The expression of cement protein genes appears to remain constant through the molt cycle and is largely confined to the sub-mantle tissue. Our results reveal a novel and potentially prominent protein to the mix of cement-related components in A. amphitrite. Despite the lack of a complete genome, sample collection allowed for extended transcriptomic analysis of pre- and post-molt barnacle samples and identified a number of highly-expressed genes. Our results highlight the complexities of this sessile marine organism as it grows via molt cycles and increases the area over which it exhibits robust adhesion to its substrate.
Topics: Animals; Computational Biology; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; High-Throughput Nucleotide Sequencing; Molecular Sequence Annotation; Molting; Proteins; Thoracica; Transcriptome
PubMed: 26496984
DOI: 10.1186/s12864-015-2076-1 -
Biotechnology and Bioengineering Feb 2018Transient gene expression (TGE) is a methodology employed in bioprocessing for the fast provision of recombinant protein material. Mild hypothermia is often introduced...
Transient gene expression (TGE) is a methodology employed in bioprocessing for the fast provision of recombinant protein material. Mild hypothermia is often introduced to overcome the low yield typically achieved with TGE and improve specific protein productivity. It is therefore of interest to examine the impact of mild hypothermic temperatures on both the yield and quality of transiently expressed proteins and the relationship to changes in cellular processes and metabolism. In this study, we focus on the ability of a Chinese hamster ovary cell line to galactosylate a recombinant monoclonal antibody (mAb) product. Through experimentation and flux balance analysis, our results show that TGE in mild hypothermic conditions led to a 76% increase in q compared to TGE at 36.5°C in our system. This increase is accompanied by increased consumption of nutrients and amino acids, together with increased production of intracellular nucleotide sugar species, and higher rates of mAb galactosylation, despite a reduced rate of cell growth. The reduction in biomass accumulation allowed cells to redistribute their energy and resources toward mAb synthesis and Fc-glycosylation. Interestingly, the higher capacity of cells to galactosylate the recombinant product in TGE at 32°C appears not to have been assisted by the upregulation of galactosyltransferases (GalTs), but by the increased expression of N-acetylglucosaminyltransferase II (GnTII) in this cell line, which facilitated the production of bi-antennary glycan structures for further processing.
Topics: Animals; Antibodies, Monoclonal; Bioreactors; CHO Cells; Cricetinae; Cricetulus; Gene Expression; Glycosylation; Immunoglobulin Fc Fragments; Metabolic Flux Analysis; Recombinant Proteins; Temperature
PubMed: 28921534
DOI: 10.1002/bit.26456 -
STAR Protocols Sep 2020By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be...
By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be acquired on the single-cell level. Here, we describe a protocol for the combined analysis of over 40 proteins and 400 genes on over 10 cells using the nano-well based Rhapsody platform. We also include a workflow for sample multiplexing, which uniquely identifies the initial source of cells (such as tissue type or donor) in the downstream analysis after upstream pooling. For complete information on the use and execution of this protocol, please refer to Mair et al. (2020).
Topics: Antibodies; Gene Expression; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Proteins; Proteomics; Sequence Analysis, RNA; Single-Cell Analysis; Transcriptome
PubMed: 33000001
DOI: 10.1016/j.xpro.2020.100092 -
ELife Oct 2021Single-cell expression profiling opens up new vistas on cellular processes. Extensive cell-to-cell variability at the transcriptomic and proteomic level has been one of...
Single-cell expression profiling opens up new vistas on cellular processes. Extensive cell-to-cell variability at the transcriptomic and proteomic level has been one of the stand-out observations. Because most experimental analyses are destructive we only have access to snapshot data of cellular states. This loss of temporal information presents significant challenges for inferring dynamics, as well as causes of cell-to-cell variability. In particular, we typically cannot separate dynamic variability from within cells ('intrinsic noise') from variability across the population ('extrinsic noise'). Here, we make this non-identifiability mathematically precise, allowing us to identify new experimental set-ups that can assist in resolving this non-identifiability. We show that multiple generic reporters from the same biochemical pathways (e.g. mRNA and protein) can infer magnitudes of intrinsic and extrinsic transcriptional noise, identifying sources of heterogeneity. Stochastic simulations support our theory, and demonstrate that 'pathway-reporters' compare favourably to the well-known, but often difficult to implement, dual-reporter method.
Topics: Gene Expression Profiling; Proteins; RNA, Messenger; Transcription, Genetic
PubMed: 34636320
DOI: 10.7554/eLife.69324 -
Infection and Immunity Aug 1998The green fluorescent protein (GFP) gene was expressed on a plasmid in B. henselae, and GFP-expressing bacteria were visualized by fluorescence microscopy. HEp-2 cells...
The green fluorescent protein (GFP) gene was expressed on a plasmid in B. henselae, and GFP-expressing bacteria were visualized by fluorescence microscopy. HEp-2 cells infected with GFP-expressing bacteria were separated from uninfected cells with a fluorescence activated cell sorter. Promoter fusions of B. henselae chromosomal DNA to gfp were examined by flow cytometry, and a B. henselae groEL promoter fusion which induced expression at 37 degreesC was isolated.
Topics: Bartonella henselae; Gene Expression; Green Fluorescent Proteins; Humans; Luminescent Proteins; Tumor Cells, Cultured
PubMed: 9673287
DOI: 10.1128/IAI.66.8.3964-3967.1998