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Physical Biology Aug 2023Cell-to-cell variability in protein concentrations is strongly affected by extrinsic noise, especially for highly expressed genes. Extrinsic noise can be due to...
Cell-to-cell variability in protein concentrations is strongly affected by extrinsic noise, especially for highly expressed genes. Extrinsic noise can be due to fluctuations of several possible cellular factors connected to cell physiology and to the level of key enzymes in the expression process. However, how to identify the predominant sources of extrinsic noise in a biological system is still an open question. This work considers a general stochastic model of gene expression with extrinsic noise represented as fluctuations of the different model rates, and focuses on the out-of-equilibrium expression dynamics. Combining analytical calculations with stochastic simulations, we characterize how extrinsic noise shapes the protein variability during gene activation or inactivation, depending on the prevailing source of extrinsic variability, on its intensity and timescale. In particular, we show that qualitatively different noise profiles can be identified depending on which are the fluctuating parameters. This indicates an experimentally accessible way to pinpoint the dominant sources of extrinsic noise using time-coarse experiments.
Topics: Cell Physiological Phenomena; Proteins; Gene Expression; Stochastic Processes; Models, Biological
PubMed: 37489881
DOI: 10.1088/1478-3975/acea4e -
The Journal of Cell Biology Feb 1998CCS embryonic stem (ES) cells possessing two mutant alleles (ry1r-/ry1r-) for the skeletal muscle ryanodine receptor (RyR) have been produced and injected subcutaneously...
CCS embryonic stem (ES) cells possessing two mutant alleles (ry1r-/ry1r-) for the skeletal muscle ryanodine receptor (RyR) have been produced and injected subcutaneously into severely compromised immunodeficient mice to produce teratocarcinomas in which Ry1R expression is absent. Several primary fibroblast cell lines were isolated and subcloned from one of these tumors that contain the knockout mutation in both alleles and exhibit a doubling time of 18-24 h, are not contact growth inhibited, do not exhibit drastic morphological change upon serum reduction, and possess the normal complement of chromosomes. Four of these fibroblast clones were infected with a retrovirus containing the cDNA encoding myoD and a puromycin selection marker. Several (1-2 microg/ml) puromycin-resistant subclones from each initial cell line were expanded and examined for their ability to express myoD and to form multinucleated myotubes that express desmin and myosin upon removal of mitogens. One of these clones (1B5 cells) was selected on this basis for further study. These cells, upon withdrawal of mitogens for 5-7 d, were shown by Western blot analysis to express key triadic proteins, including skeletal triadin, calsequestrin, FK506-binding protein, 12 kD, sarco(endo)plasmic reticulum calcium-ATPase1, and dihydropyridine receptors. Neither RyR isoform protein, Ry1R (skeletal), Ry2R (cardiac), nor Ry3R (brain), were detected in differentiated 1B5 cells. Measurements of intracellular Ca2+ by ratio fluorescence imaging of fura-2-loaded cells revealed that differentiated 1B5 cells exhibited no responses to K+ (40 mM) depolarization, ryanodine (50-500 microM), or caffeine (20-100 mM). Transient transfection of the 1B5 cells with the full-length rabbit Ry1R cDNA restored the expected responses to K+ depolarization, caffeine, and ryanodine. Depolarization-induced Ca2+ release was independent of extracellular Ca2+, consistent with skeletal-type excitation-contraction coupling. Wild-type Ry1R expressed in 1B5 cells were reconstituted into bilayer lipid membranes and found to be indistinguishable from channels reconstituted from rabbit sarcoplasmic reticulum with respect to unitary conductance, open dwell times, and responses to ryanodine and ruthenium red. The 1B5 cell line provides a powerful and easily managed homologous expression system in which to study how Ry1R structure relates to function.
Topics: Animals; Calcium Channels; Cell Line; DNA, Complementary; Gene Expression; Genetic Engineering; Mice; Mice, Inbred Strains; Mice, Mutant Strains; Muscle Proteins; Muscle, Skeletal; Mutation; Recombinant Proteins; Ryanodine Receptor Calcium Release Channel; Stem Cells; Transfection; Transgenes
PubMed: 9472036
DOI: 10.1083/jcb.140.4.843 -
Journal of Korean Medical Science Aug 2005Defensins and cathelicidins (LL-37) are major antimicrobial peptides (AMPs) of the innate immune system of the human skin. In normal non-inflamed skin these peptides are... (Comparative Study)
Comparative Study
Defensins and cathelicidins (LL-37) are major antimicrobial peptides (AMPs) of the innate immune system of the human skin. In normal non-inflamed skin these peptides are negligible, but their expression can be markedly increased in inflammatory skin disease such as psoriasis. We designed this study to identify the expressions of LL-37 in normal human keratinocyte (NHK) and HaCaT cells after exposure to stimulants and to investigate difference of LL-37 expression accompanied with cell differentiation status, and come to understand difference of susceptibility to infection in atopic dermatitis and psoriasis. Expressions of LL-37 in NHKs and HaCaT cells were evaluated by using RT-PCR, Western blotting, and immunohistochemical (IHC) staining at 6, 12, and 24 hr post stimulation after exposure to Ultraviolet B irradiation and lipopolysaccharide. And expression of LL-37 in skin biopsy specimens from patients with atopic dermatitis and psoriasis was determined by immunohistochemical analysis. In time-sequential analyses of LL-37 expression revealed that LL-37 was expressed in NHKs, but not in HaCaT cells. IHC analysis confirmed the presence of abundant LL-37 in the epidermis of psoriasis. Therefore we deduced that expression of LL-37 is affected by UV irradiation, bacterial infection, and status of cell differentiation.
Topics: Antimicrobial Cationic Peptides; Blotting, Western; Cell Line; Cells, Cultured; Defensins; Dose-Response Relationship, Drug; Gene Expression; Humans; Immunohistochemistry; Keratinocytes; Lipopolysaccharides; Male; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Skin; Skin Diseases; Cathelicidins
PubMed: 16100459
DOI: 10.3346/jkms.2005.20.4.649 -
Virology Oct 1996The expression kinetics of an essential trans-regulatory protein, ICP27, from herpes simplex virus type 1 correspond to that of an immediate-early gene whose expression...
The expression kinetics of an essential trans-regulatory protein, ICP27, from herpes simplex virus type 1 correspond to that of an immediate-early gene whose expression increases rapidly upon infection and then decreases as of 7 hr postinfection. In contrast, here we report that the bovine herpesvirus type 1 (BHV-1) homolog BICP27, a 50-kDa protein, is expressed as an early gene. Both the transcript and protein accumulated gradually reaching peak levels at approximately 12 hr postinfection, after which point steady state levels were maintained up to 24 hr. Thus the expression profiles of ICP27 and BICP27 are significantly different, suggesting that they may possess different functions.
Topics: Animals; COS Cells; Cattle; Gene Expression; Herpesvirus 1, Bovine; Humans; Immediate-Early Proteins; Kinetics; Phosphoproteins; Protein Biosynthesis; Transcription, Genetic; Viral Proteins
PubMed: 8862429
DOI: 10.1006/viro.1996.0536 -
PloS One Feb 2011Structural and biophysical studies of protein complexes require multi-milligram quantities of soluble material. Subunits are often unstable when expressed separately so...
Structural and biophysical studies of protein complexes require multi-milligram quantities of soluble material. Subunits are often unstable when expressed separately so co-expression strategies are commonly employed since in vivo complex formation can provide stabilising effects. Defining constructs for subunit co-expression experiments is difficult if the proteins are poorly understood. Even more problematic is when subunit polypeptide chains co-fold since individually they do not have predictable domains. We have developed CoESPRIT, a modified version of the ESPRIT random library construct screen used previously on single proteins, to express soluble protein complexes. A random library of target constructs is screened against a fixed bait protein to identify stable complexes. In a proof-of-principle study, C-terminal fragments of the influenza polymerase PB2 subunit containing folded domains were isolated using importin alpha as bait. Separately, a C-terminal fragment of the PB1 subunit was used as bait to trap N-terminal fragments of PB2 resulting in co-folded complexes. Subsequent expression of the target protein without the bait indicates whether the target is independently stable, or co-folds with its partner. This highly automated method provides an efficient strategy for obtaining recombinant protein complexes at yields compatible with structural, biophysical and functional studies.
Topics: Amino Acid Sequence; Animals; Cloning, Molecular; Gene Expression; Gene Library; Gene Regulatory Networks; High-Throughput Screening Assays; Humans; Models, Biological; Models, Molecular; Multiprotein Complexes; Plasmids; Protein Array Analysis; Recombinant Proteins; Research Design; Solubility
PubMed: 21364980
DOI: 10.1371/journal.pone.0016261 -
Developmental Dynamics : An Official... May 2005The reticulon (RTN) family of proteins has been described as a new eukaryotic protein family. We have isolated Xenopus cDNA homologues of RTN2 and RTN3 and examined...
The reticulon (RTN) family of proteins has been described as a new eukaryotic protein family. We have isolated Xenopus cDNA homologues of RTN2 and RTN3 and examined their expression patterns during Xenopus development. XRTN2 has two transcripts, XRTN2-B and XRTN2-C, which encode 321 and 191 amino acids, respectively. XRTN3 has only one transcript that encodes 214 amino acids. We detected the XRTN2-B transcript in the neural tissues and brain from the early neurula stage. XRTN2-C is strongly expressed in the myotome, future skeletal muscle. The XRTN3 mRNA is localized in the animal hemisphere of the egg and blastula stage embryos and then subsequently restricted, mainly in the neural tissues. At the subcellular level, The XRTN proteins are expressed in the endoplasmic reticulum network structure of the animal cap cells as well as COS-7 cells. Our results suggest the potential roles of XRTN2s and XRTN3 during Xenopus embryogenesis.
Topics: Amino Acid Sequence; Animals; Embryo, Nonmammalian; Endoplasmic Reticulum; Gene Expression; Humans; Mice; Molecular Sequence Data; Nerve Tissue Proteins; Sequence Alignment; Sequence Analysis, Protein; Xenopus Proteins; Xenopus laevis
PubMed: 15765506
DOI: 10.1002/dvdy.20327 -
Scientific Reports Oct 2020With considerable accumulation of RNA-Seq transcriptome data, we have extended our understanding about protein-coding gene transcript compositions. However,...
With considerable accumulation of RNA-Seq transcriptome data, we have extended our understanding about protein-coding gene transcript compositions. However, alternatively compounded patterns of human protein-coding gene transcripts would complicate gene expression data processing and interpretation. It is essential to exhaustively interrogate complex mRNA isoforms of protein-coding genes with an unified data resource. In order to investigate representative mRNA transcript isoforms to be utilized as transcriptome analysis references, we utilized GTEx data to establish a top-ranked transcript isoform expression data resource for human protein-coding genes. Distinctive tissue specific expression profiles and modulations could be observed for individual top-ranked transcripts of protein-coding genes. Protein-coding transcripts or genes do occupy much higher expression fraction in transcriptome data. In addition, top-ranked transcripts are the dominantly expressed ones in various normal tissues. Intriguingly, some of the top-ranked transcripts are noncoding splicing isoforms, which imply diverse gene regulation mechanisms. Comprehensive investigation on the tissue expression patterns of top-ranked transcript isoforms is crucial. Thus, we established a web tool to examine top-ranked transcript isoforms in various human normal tissue types, which provides concise transcript information and easy-to-use graphical user interfaces. Investigation of top-ranked transcript isoforms would contribute understanding on the functional significance of distinctive alternatively spliced transcript isoforms.
Topics: Datasets as Topic; Gene Expression; Gene Expression Profiling; Genes; Humans; Proteins; Transcriptome
PubMed: 33004865
DOI: 10.1038/s41598-020-73081-5 -
The Korean Journal of Parasitology Oct 2020Clonorchis sinensis is a food-borne trematode that infects more than 15 million people. The liver fluke causes clonorchiasis and chronical cholangitis, and promotes...
Clonorchis sinensis is a food-borne trematode that infects more than 15 million people. The liver fluke causes clonorchiasis and chronical cholangitis, and promotes cholangiocarcinoma. The underlying molecular pathogenesis occurring in the bile duct by the infection is little known. In this study, transcriptome profile in the bile ducts infected with C. sinensis were analyzed using microarray methods. Differentially expressed genes (DEGs) were 1,563 and 1,457 at 2 and 4 weeks after infection. Majority of the DEGs were temporally dysregulated at 2 weeks, but 519 DEGs showed monotonically changing expression patterns that formed seven distinct expression profiles. Protein-protein interaction (PPI) analysis of the DEG products revealed 5 sub-networks and 10 key hub proteins while weighted co-expression network analysis (WGCNA)-derived gene-gene interaction exhibited 16 co-expression modules and 13 key hub genes. The DEGs were significantly enriched in 16 Kyoto Encyclopedia of Genes and Genomes pathways, which were related to original systems, cellular process, environmental information processing, and human diseases. This study uncovered a global picture of gene expression profiles in the bile ducts infected with C. sinensis, and provided a set of potent predictive biomarkers for early diagnosis of clonorchiasis.
Topics: Animals; Bile Ducts; Clonorchiasis; Clonorchis sinensis; Early Diagnosis; Epistasis, Genetic; Gene Expression; Helminth Proteins; Rats, Sprague-Dawley; Signal Transduction; Transcriptome
PubMed: 33202503
DOI: 10.3347/kjp.2020.58.5.513 -
PloS One 2011Efficient and stoichiometric expression of genes concatenated by bi- or multi-cistronic vectors has become an invaluable tool not only in basic biology to track and...
Efficient and stoichiometric expression of genes concatenated by bi- or multi-cistronic vectors has become an invaluable tool not only in basic biology to track and visualize proteins in vivo, but also for vaccine development and in the clinics for gene therapy. To adequately compare, in vivo, the effectiveness of two of the currently popular co-expression strategies - the internal ribosome entry site (IRES) derived from the picornavirus and the 2A peptide from the foot-and-mouth disease virus (FDMV) (F2A), we analyzed two locus-specific knock-in mouse lines co-expressing SRY-box containing gene 9 (Sox9) and enhanced green fluorescent protein (EGFP) linked by the IRES (Sox9(IRES-EGFP)) or the F2A (Sox9(F2A-EGFP)) sequence. Both the constructs expressed Sox9 and EGFP proteins in the appropriate Sox9 expression domains, with the IRES construct expressing reduced levels of EGFP compared to that of the F2A. The latter, on the other hand, produced about 42.2% Sox9-EGFP fusion protein, reflecting an inefficient ribosome 'skipping' mechanism. To investigate if the discrepancy in the 'skipping' process was locus-dependent, we further analyzed the FLAG(3)-Bapx1(F2A-EGFP) mouse line and found similar levels of fusion protein being produced. To assess if EGFP was hindering the 'skipping' mechanism, we examined another mouse line co-expressing Bagpipe homeobox gene 1 homolog (Bapx1), Cre recombinase and EGFP (Bapx1(F2A-Cre-F2A-EGFP)). While the 'skipping' was highly efficient between Bapx1 and Cre, the 'skipping' between Cre and EGFP was highly inefficient. We have thus demonstrated in our comparison study that the efficient and close to equivalent expression of genes linked by F2A is achievable in stable mouse lines, but the EGFP reporter may cause undesirable inhibition of the 'skipping' at the F2A sequence. Hence, the use of other reporter genes should be explored when utilizing F2A peptides.
Topics: Animals; Flow Cytometry; Foot-and-Mouth Disease Virus; Gene Expression; Green Fluorescent Proteins; Mice; Ribosomes; Viral Proteins
PubMed: 22216134
DOI: 10.1371/journal.pone.0028885 -
Biology of Sex Differences Aug 2017Pre-implantation embryos exhibit sexual dimorphisms in both primates and rodents. To determine whether these differences reflected sex-biased expression patterns, we...
BACKGROUND
Pre-implantation embryos exhibit sexual dimorphisms in both primates and rodents. To determine whether these differences reflected sex-biased expression patterns, we generated transcriptome profiles for six 40,XX, six 40,XY, and two 39,X mouse embryonic stem (ES) cells by RNA sequencing.
RESULTS
We found hundreds of coding and non-coding RNAs that were differentially expressed between male and female cells. Surprisingly, the majority of these were autosomal and included RNA encoding transcription and epigenetic and chromatin remodeling factors. We showed differential Prdm14-responsive enhancer activity in male and female cells, correlating with the sex-specific levels of Prdm14 expression. This is the first time sex-specific enhancer activity in ES cells has been reported. Evaluation of X-linked gene expression patterns between our XX and XY lines revealed four distinct categories: (1) genes showing 2-fold greater expression in the female cells; (2) a set of genes with expression levels well above 2-fold in female cells; (3) genes with equivalent RNA levels in male and female cells; and strikingly, (4) a small number of genes with higher expression in the XY lines. Further evaluation of autosomal gene expression revealed differential expression of imprinted loci, despite appropriate parent-of-origin patterns. The 39,X lines aligned closely with the XY cells and provided insights into potential regulation of genes associated with Turner syndrome in humans. Moreover, inclusion of the 39,X lines permitted three-way comparisons, delineating X and Y chromosome-dependent patterns.
CONCLUSIONS
Overall, our results support the role of the sex chromosomes in establishing sex-specific networks early in embryonic development and provide insights into effects of sex chromosome aneuploidies originating at those stages.
Topics: Animals; Cell Line; DNA-Binding Proteins; Embryonic Stem Cells; Female; Gene Expression; Gene Expression Regulation, Developmental; Genetic Loci; Male; Mice, Inbred C57BL; Promoter Regions, Genetic; RNA; RNA-Binding Proteins; Sex Characteristics; Sex Chromosomes; Transcription Factors
PubMed: 28818098
DOI: 10.1186/s13293-017-0150-x