scholarly journals Reconstitution of an intact clock that generates circadian DNA binding in vitro

2020 ◽  
Author(s):  
Archana G. Chavan ◽  
Dustin C. Ernst ◽  
Mingxu Fang ◽  
Cigdem Sancar ◽  
Carrie L. Partch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Circadian Clock ◽  
Real Time ◽  
Clock System ◽  
Regulatory Circuit ◽  
Time Observation ◽  
Promoter Dna ◽  
High Throughput Assay ◽  
User Intervention

AbstractCircadian clocks control gene expression in the complex milieu of cells. Here, we reconstituted under defined conditions in vitro the cyanobacterial circadian clock system which includes an oscillator, signal-transduction pathways, transcription factor, and promoter DNA. The system oscillates autonomously with a near 24 h period, remains phase coherent for many days, and allows real-time observation of each component simultaneously without user intervention. This reassembled clock system provides new insights into how a circadian clock exerts control over gene expression and can serve in the area of synthetic biology as a new platform upon which to build even more complexity.One Sentence SummaryAn autonomously oscillating circadian clock-controlled gene regulatory circuit is studied in vitro using a real-time high-throughput assay.

scholarly journals ZipSeq : Barcoding for Real-time Mapping of Single Cell Transcriptomes

2020 ◽  
Author(s):  
Kenneth H. Hu ◽  
John P. Eichorst ◽  
Chris S. McGinnis ◽  
David M. Patterson ◽  
Eric D. Chow ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Real Time ◽  
Dimensional Space ◽  
Single Cells ◽  
Expression Patterns ◽  
Live Cells ◽  
Glass Substrates ◽  
Complete Mapping

ABSTRACTSpatial transcriptomics seeks to integrate single-cell transcriptomic data within the 3-dimensional space of multicellular biology. Current methods use glass substrates pre-seeded with matrices of barcodes or fluorescence hybridization of a limited number of probes. We developed an alternative approach, called ‘ZipSeq’, that uses patterned illumination and photocaged oligonucleotides to serially print barcodes (Zipcodes) onto live cells within intact tissues, in real-time and with on-the-fly selection of patterns. Using ZipSeq, we mapped gene expression in three settings: in-vitro wound healing, live lymph node sections and in a live tumor microenvironment (TME). In all cases, we discovered new gene expression patterns associated with histological structures. In the TME, this demonstrated a trajectory of myeloid and T cell differentiation, from periphery inward. A variation of ZipSeq efficiently scales to the level of single cells, providing a pathway for complete mapping of live tissues, subsequent to real-time imaging or perturbation.

Evolution Analysis of the Circadian Clock Protein KaiB

2013 ◽  
Vol 647 ◽  
pp. 391-395
Author(s):  
Liu Sen ◽  
Song Liu
Keyword(s):  
Circadian Rhythms ◽  
Circadian Clock ◽  
Feedback Loop ◽  
Circadian System ◽  
Circadian Rhythmicity ◽  
Physiological Functions ◽  
Clock System ◽  
Evolution Analysis ◽  
Clock Protein

Regulation of daily physiological functions with approximate a 24-hour periodicity, or circadian rhythms, is a characteristic of eukaryotes. So far, cyanobacteria are only known prokaryotes reported to possess circadian rhythmicity. The circadian system in cyanobacteria comprises both a post-translational oscillator (PTO) and a transcriptional/translational feedback loop (TTFL). The PTO can be reconstituted in vitro with three purified proteins (KaiA, KaiB, and KaiC) with the existence of ATP. Phase of the nanoclockwork has been associated with the phosphorylation states of KaiC, with KaiA promoting the phosphorylation of KaiC, and KaiB de-phosphorylating KaiC. Here we studied the evolution of the KaiB protein. The result will be helpful in understanding the evolution of the circadian clock system.

Analyzing Gene Expression through Real Time PCR while Neo-tissue Regeneration using Developed Tissue Constructs

2020 ◽  
pp. 15-34
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Tissue Regeneration ◽  
Real Time Pcr ◽  
Molecular Level ◽  
Northern Blotting ◽  
Tissue Constructs ◽  
Low Sensitivity

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

209 9-cis RETINOIC ACID INHIBITS EXPRESSION OF AKR1B1 TRANSCRIPT IN THE BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 252
Author(s):  
G. K. Deb ◽  
S. R. Dey ◽  
K. S. Huque ◽  
M. Fokruzzaman ◽  
K. L. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Embryo Implantation ◽  
Cumulus Cell ◽  
Prostaglandin F2α ◽  
Receptor Expression ◽  
Bovine Embryo

Quantitative real-time PCR has enabled quality evaluation of oocyte and pre-implantation embryo through monitoring expression of several molecular markers that are involved in metabolic activity, stress response, reprogramming, and other biological events. The aldo-keto reductase family 1 member B1 (AKR1B1) transcript is potentially involved in pregnancy failure through metabolism of progesterone and synthesis of prostaglandin F2α in the bovine uterine endometrium. High expression of the transcript in blastocysts correlates inhibition of embryo implantation and/or embryo resorption. Maturation of immature oocyte in presence of 9-cis retinoic acid (9-cis RA) increases in vitro bovine embryo development rates and embryo quality. These beneficial effects of 9-cis RA are mediated through multiple mechanisms, including FSH/LH receptor expression, polyadenylation, growth factor signalling, oxidative-stress protection, or decreasing oocyte TNFα gene expression and inhibiting cumulus cell apoptosis during maturation. The present study aimed to evaluate the effect of 9-cis RA on expression pattern of AKR1B1 transcript in the oocyte matured in vitro and embryos (8-cell and Day 8 blastocyst) produced from in vitro matured oocytes in presence or absence of 9-cis RA. Bovine cumulus–oocyte complexes, isolated from ovaries collected at the abattoir, were matured in vitro in the presence of zero (control) or 5 nM 9-cis RA in the maturation medium (TCM199 + 10% fetal bovine serum + 1 µg mL–1 β-oestradiol + 10 µg mL–1 follicle stimulating hormone + 0.6 mM cystein and 0.2 mM Na-pyruvate). After maturation, the oocytes were subjected to standardized in vitro embryo production protocol or oocyte samples were collected for gene expression analysis. The expression of AKR1B1 transcript was quantified in zona-free oocytes, 8-cell embryos, and Day 8 blastocysts by real-time PCR using SYBER green. Not less than 4 biological replicates (oocytes: 50 to 60 per replicate and 8-cell embryos/day-8 blastocyst: 3 to 5 per replicate) were done for each group. The expression was normalized against a minimum of 2 out of 4 reference transcripts (18S rRNA, β-actin, glyceraldehyde-3-phosphate dehydrogenase and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) analysed each time with AKR1B1. The best combination of reference genes was automatically calculated by the CFX manager V1.1 program (Bio-Rad) based on M-value. The differences in gene expression levels were tested by Student’s t-test. Results indicated that 9-cis RA decreased expression of AKR1B1 transcript in the oocyte (1.0- v. 2.0-fold; P < 0.05), 8-cell-embryos (1.0- v. 10.1-fold; P < 0.03), and blastocyst (1.0- v. 2.1-fold; P < 0.03) compared with control. In conclusion, the present study indicates that 9-cis RA inhibits AKR1B1 transcript expression in oocytes and pre-implantation embryos.

scholarly journals Real-Time In Vitro Fluorescence Anisotropy of the Cyanobacterial Circadian Clock

2019 ◽  
Vol 2 (2) ◽  
pp. 42 ◽  
Author(s):  
Joel Heisler ◽  
Archana Chavan ◽  
Yong-Gang Chang ◽  
Andy LiWang
Keyword(s):  
Circadian Clock ◽  
Real Time ◽  
Fluorescence Anisotropy ◽  
Polyacrylamide Gel Electrophoresis ◽  
Live Cells ◽  
Ex Post ◽  
Clock Proteins ◽  
Ex Post Facto ◽  
Clock Reaction

Uniquely, the circadian clock of cyanobacteria can be reconstructed outside the complex milieu of live cells, greatly simplifying the investigation of a functioning biological chronometer. The core oscillator component is composed of only three proteins, KaiA, KaiB, and KaiC, and together with ATP they undergo waves of assembly and disassembly that drive phosphorylation rhythms in KaiC. Typically, the time points of these reactions are analyzed ex post facto by denaturing polyacrylamide gel electrophoresis, because this technique resolves the different states of phosphorylation of KaiC. Here, we describe a more sensitive method that allows real-time monitoring of the clock reaction. By labeling one of the clock proteins with a fluorophore, in this case KaiB, the in vitro clock reaction can be monitored by fluorescence anisotropy on the minutes time scale for weeks.

scholarly journals Developmental- and differentiation-specific patterns of human γ- and β-globin promoter DNA methylation

Blood ◽  
2007 ◽  
Vol 110 (4) ◽  
pp. 1343-1352 ◽  
Author(s):  
Rodwell Mabaera ◽  
Christine A. Richardson ◽  
Kristin Johnson ◽  
Mei Hsu ◽  
Steven Fiering ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Promoter Methylation ◽  
Fetal Liver ◽  
Globin Gene ◽  
Erythroid Cells ◽  
Promoter Dna Methylation ◽  
Globin Promoter ◽  
Promoter Dna

AbstractThe mechanisms underlying the human fetal-to-adult β-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human γ- and β-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at −162 of the γ promoter and −126 of the β promoter are hypomethylated in ABM and FL, respectively. We also studied γ-globin promoter methylation during in vitro differentiation of erythroid cells. The γ promoters are initially hypermethylated in CD34+ cells. The upstream γ promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient γ-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human γ- and β-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human β-globin locus gene switching.

scholarly journals The disruption of circadian clockwork in differentiating cells from rat reproductive tissues as identified by in vitro real-time monitoring system

2007 ◽  
Vol 193 (3) ◽  
pp. 413-420 ◽  
Author(s):  
Pei-Jian He ◽  
Masami Hirata ◽  
Nobuhiko Yamauchi ◽  
Seiichi Hashimoto ◽  
Masa-aki Hattori
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Monitoring System ◽  
Cell Types ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Peripheral Tissues ◽  
Reproductive Tissues ◽  
Clock Gene Expression

The circadian clock, regulating hormonal secretion and metabolisms in accordance with the environmental light–dark cycle, resides in almost all peripheral tissues as well as in the superchiasmatic nucleus. Clock gene expression has been found to be noncyclic during spermatogenesis and the differentiation of thymocytes. However, currently little is known about how cell differentiation could affect circadian clockwork. We performed this study using the in vitro real-time oscillation monitoring system to examine the clockwork in several types of differentiating cells originated from reproductive tissues of transgenic rats (constructed with Period gene 2 (Per2) promoter-destabilized luciferase reporter gene). After treatment with dexamethasone (DXM), persistent oscillation of Per2 expression was observed in both gonadotropin-induced and pregnant ovarian luteal cells, proliferative uterine stromal cells (USCs), and nondifferentiating testicular interstitial cells, with a cyclic period of ~24 h. In contrast to these cell types, only one cycle of oscillation was sustained in granulosa cells undergoing differentiation. Additionally, Per2 oscillation was irregular in USCs undergoing decidualization induced by medroxyprogesterone acetate plus N6, 2-O-dibutyryl adenosine 3′:5′-cyclic monophosphate. Furthermore, no oscillation of Per2 expression was evoked by DXM in Leydig cells and thymocytes. In conclusion, the present study characterized the oscillation of Per2 gene expression in several types of ovarian, uterine, and testicular cells, and it is strongly suggested that circadian clockwork is affected during cellular differentiation.

scholarly journals Global H-NS counter-silencing by LuxR activates quorum sensing gene expression

2019 ◽  
Author(s):  
Ryan R Chaparian ◽  
Minh L N Tran ◽  
Laura C Miller Conrad ◽  
Douglas B Rusch ◽  
Julia C van Kessel
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Cell Communication ◽  
Binding Activity ◽  
Primary Role ◽  
Dna Binding Activity ◽  
Cellular Behaviors ◽  
A Cell ◽  
Promoter Dna

Abstract Bacteria coordinate cellular behaviors using a cell–cell communication system termed quorum sensing. In Vibrio harveyi, the master quorum sensing transcription factor LuxR directly regulates >100 genes in response to changes in population density. Here, we show that LuxR derepresses quorum sensing loci by competing with H-NS, a global transcriptional repressor that oligomerizes on DNA to form filaments and bridges. We first identified H-NS as a repressor of bioluminescence gene expression, for which LuxR is a required activator. In an hns deletion strain, LuxR is no longer necessary for transcription activation of the bioluminescence genes, suggesting that the primary role of LuxR is to displace H-NS to derepress gene expression. Using RNA-seq and ChIP-seq, we determined that H-NS and LuxR co-regulate and co-occupy 28 promoters driving expression of 63 genes across the genome. ChIP-PCR assays show that as autoinducer concentration increases, LuxR protein accumulates at co-occupied promoters while H-NS protein disperses. LuxR is sufficient to evict H-NS from promoter DNA in vitro, which is dependent on LuxR DNA binding activity. From these findings, we propose a model in which LuxR serves as a counter-silencer at H-NS-repressed quorum sensing loci by disrupting H-NS nucleoprotein complexes that block transcription.

Gene Expression Profiling in Multiple Myeloma Cells Treated with the Novel Anti-Myeloma Agents Zoledronate and Fluvastatin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5078-5078
Author(s):  
Timothy J. Molloy ◽  
Baulch-Brown Cindy ◽  
Yi-Mo Deng ◽  
Andrew Spencer ◽  
David F. Ma
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Real Time ◽  
Real Time Pcr ◽  
Mevalonate Pathway ◽  
Annexin V ◽  
Quantitative Real Time Pcr ◽  
Myeloma Cells ◽  
Qrt Pcr

Abstract We have shown in vitro that multiple myeloma (MM) cells can be destroyed by treating them with the mevalonate pathway inhibitors zoledronate and fluvastatin. While the efficacy of these compounds singly and combination have been demonstrated, their exact modes of action remain largely unknown. The present study aimed to use microarray and quantitative real-time PCR (QRT-PCR) techniques to analyse gene expression in treated myeloma cells to identify novel genes and pathways involved in the anti-myeloma action of these compounds. The human MM cell line NCI-H929 was treated with zoledronate and fluvastatin singly and in combination, and RNA was extracted and used to interrogate oligonucleotide microarrays consisting of 19,000 features representing known and unknown genes. Quantitative real-time PCR was subsequently used to confirm the expression of several genes of interest. Flow cytometry with Annexin V FITC staining was used to detect apoptosis. It was observed that genes related to apoptosis (caspases and p53-related genes), cell cycle control (cyclins), GTPase signalling (Rabs), and growth and proliferation (growth factors) were particularly affected by zoledronate and fluvastatin, and some of these genetic effects were synergistic when a combination of zoledronate and fluvastatin was used. QRT-PCR confirmed the effects on the caspase- and p53-related apoptotic pathways, and these effects were correlated with increased apoptosis in the myeloma cells. The mevalonate pathway inhibitors fluvastatin and zoledronate are highly efficient at killing MM cells, and their effects appear to be synergistic. Our microarray and QT-PCR analyses demonstrated that the expression of specific groups of genes important to the survival and proliferation of myeloma cells are affected by these compounds. p53 and caspase-dependent pathways appear to be the key apoptotic cascades stimulated. Insights into the mechanisms of these novel therapeutics are important as they might help to define their roles in the treatment of multiple myeloma.

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