scholarly journals Spatial control of gene expression in flies using bacterially derived binary transactivation systems

2020 ◽  
Author(s):  
Stephanie Gamez ◽  
Luis C. Vesga ◽  
Stelia C. Mendez-Sanchez ◽  
Omar S. Akbari
Keyword(s):  
Gene Expression ◽  
Pseudomonas Putida ◽  
Caulobacter Crescentus ◽  
Streptomyces Coelicolor ◽  
Molecular Genetic ◽  
Genetic Studies ◽  
Control Of Gene Expression ◽  
Multiple Systems

AbstractControlling gene expression is an instrumental tool for biotechnology, as it enables the dissection of gene function, affording precise spatial-temporal resolution. To generate this control, binary transactivational systems have been used employing a modular activator consisting of a DNA binding domain(s) fused to activation domain(s). For fly genetics, many binary transactivational systems have been exploited in vivo; however as the study of complex problems often requires multiple systems that can be used in parallel, there is a need to identify additional bipartite genetic systems. To expand this molecular genetic toolbox, we tested multiple bacterially-derived binary transactivational systems in Drosophila melanogaster including the p-CymR operon from Pseudomonas putida, PipR operon from Streptomyces coelicolor, TtgR operon from Pseudomonas putida, and the VanR operon from Caulobacter crescentus. Our work provides the first characterization of these systems in an animal model in vivo. For each system we demonstrate robust tissue-specific spatial transactivation of reporter gene expression, enabling future studies to exploit these transactivational systems for molecular genetic studies.

scholarly journals Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

2021 ◽  
Vol 4 (1) ◽  
pp. 22
Author(s):  
Mrinmoyee Majumder ◽  
Viswanathan Palanisamy
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Control Of Gene Expression ◽  
Regulatory Pathways ◽  
Advantages And Disadvantages ◽  
Protein Nucleic Acid

Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

scholarly journals Computational design and experimental characterization of a photo-controlled mRNA-cap guanine-N7 methyltransferase

2021 ◽  
Author(s):  
Dennis Reichert ◽  
Helena Schepers ◽  
Julian Simke ◽  
Horst Lechner ◽  
Wolfgang Dörner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Computational Design ◽  
Eukaryotic Cells ◽  
Transcriptional Level ◽  
Experimental Characterization ◽  
Temporal Control ◽  
Control Of Gene Expression ◽  
Multicellular Organisms

The spatial and temporal control of gene expression at the post-transcriptional level is essential in eukaryotic cells and developing multicellular organisms. In recent years optochemical and optogenetic tools have enabled...

Abstract 286: Long Noncoding RNAs Are Differentially Expressed in Heart Failure

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Emma L Robinson ◽  
Syed Haider ◽  
Hillary Hei ◽  
Richard T Lee ◽  
Roger S Foo
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Significant Proportion ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Control Of Gene Expression ◽  
Failing Heart ◽  
Novel Transcripts ◽  
Non Coding Rnas

Heart failure comprises of clinically distinct inciting causes but a consistent pattern of change in myocardial gene expression supports the hypothesis that unifying biochemical mechanisms underlie disease progression. The recent RNA-seq revolution has enabled whole transcriptome profiling, using deep-sequencing technologies. Up to 70% of the genome is now known to be transcribed into RNA, a significant proportion of which is long non-coding RNAs (lncRNAs), defined as polyribonucleotides of ≥200 nucleotides. This project aims to discover whether the myocardium expression of lncRNAs changes in the failing heart. Paired end RNA-seq from a 300-400bp library of ‘stretched’ mouse myocyte total RNA was carried out to generate 76-mer sequence reads. Mechanically stretching myocytes with equibiaxial stretch apparatus mimics pathological hypertrophy in the heart. Transcripts were assembled and aligned to reference genome mm9 (UCSC), abundance determined and differential expression of novel transcripts and alternative splice variants were compared with that of control (non-stretched) mouse myocytes. Five novel transcripts have been identified in our RNA-seq that are differentially expressed in stretched myocytes compared with non-stretched. These are regions of the genome that are currently unannotated and potentially are transcribed into non-coding RNAs. Roles of known lncRNAs include control of gene expression, either by direct interaction with complementary regions of the genome or association with chromatin remodelling complexes which act on the epigenome.Changes in expression of genes which contribute to the deterioration of the failing heart could be due to the actions of these novel lncRNAs, immediately suggesting a target for new pharmaceuticals. Changes in the expression of these novel transcripts will be validated in a larger sample size of stretched myocytes vs non-stretched myocytes as well as in the hearts of transverse aortic constriction (TAC) mice vs Sham (surgical procedure without the aortic banding). In vivo investigations will then be carried out, using siLNA antisense technology to silence novel lncRNAs in mice.

scholarly journals Effect of destrin mutations on the gene expression profile in vivo

2008 ◽  
Vol 34 (1) ◽  
pp. 9-21 ◽  
Author(s):  
Angela M. Verdoni ◽  
Natsuyo Aoyama ◽  
Akihiro Ikeda ◽  
Sakae Ikeda
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Target Genes ◽  
Actin Dynamics ◽  
In Vivo Model ◽  
Strong Impact ◽  
Filamentous Actin ◽  
Control Of Gene Expression

Remodeling of the actin cytoskeleton through actin dynamics (assembly and disassembly of filamentous actin) is known to be essential for numerous basic biological processes. In addition, recent studies have provided evidence that actin dynamics participate in the control of gene expression. A spontaneous mouse mutant, corneal disease 1 ( corn1), is deficient for a regulator of actin dynamics, destrin (DSTN, also known as ADF), which causes epithelial hyperproliferation and neovascularization in the cornea. Dstn corn1 mice exhibit an actin dynamics defect in the corneal epithelial cells, offering an in vivo model to investigate cellular mechanisms affected by the Dstn mutation and resultant actin dynamics abnormalities. To examine the effect of the Dstn corn1 mutation on the gene expression profile, we performed a microarray analysis using the cornea from Dstn corn1 and wild-type mice. A dramatic alteration of the gene expression profile was observed in the Dstn corn1 cornea, with 1,226 annotated genes differentially expressed. Functional annotation of these genes revealed that the most significantly enriched functional categories are associated with actin and/or cytoskeleton. Among genes that belong to these categories, a considerable number of serum response factor target genes were found, indicating the possible existence of an actin-SRF pathway of transcriptional regulation in vivo. A comparative study using an allelic mutant strain with milder corneal phenotypes suggested that the level of filamentous actin may correlate with the level of gene expression changes. Our study shows that Dstn mutations and resultant actin dynamics abnormalities have a strong impact on the gene expression profile in vivo.

Cell biology of transcription and pre-mRNA splicing: nuclear architecture meets nuclear function

2000 ◽  
Vol 113 (11) ◽  
pp. 1841-1849 ◽  
Author(s):  
T. Misteli
Keyword(s):  
Gene Expression ◽  
Cell Nucleus ◽  
Cell Biology ◽  
Nuclear Architecture ◽  
Molecular Techniques ◽  
Mrna Splicing ◽  
Cellular Organization ◽  
Control Of Gene Expression ◽  
Expression Of Genes

Gene expression is a fundamental cellular process. The basic mechanisms involved in expression of genes have been characterized at the molecular level. A major challenge is now to uncover how transcription, RNA processing and RNA export are organized within the cell nucleus, how these processes are coordinated with each other and how nuclear architecture influences gene expression and regulation. A significant contribution has come from cell biological approaches, which combine molecular techniques with microscopy methods. These studies have revealed that the mammalian cell nucleus is a complex but highly organized organelle, which contains numerous subcompartments. I discuss here how two essential nuclear processes - transcription and pre-mRNA splicing - are spatially organized and coordinated in vivo, and how this organization might contribute to the control of gene expression. The dynamic nature of nuclear proteins and compartments indicates a high degree of plasticity in the cellular organization of nuclear functions. The cellular organization of transcription and splicing suggest that the morphology of nuclear compartments is largely determined by the activities of the nucleus.

scholarly journals Red flour beetle (Tribolium castaneum): From population genetics to functional genomics

2018 ◽  
pp. 1043-1046 ◽  
Author(s):  
Harshit Kumar ◽  
Manjit Panigrahi ◽  
Supriya Chhotaray ◽  
V. Bhanuprakash ◽  
Rahul Shandilya ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Tribolium Castaneum ◽  
Insertional Mutagenesis ◽  
Molecular Genetic ◽  
Genetic Studies ◽  
Immune Priming ◽  
Systemic Rnai ◽  
Basic Facts

Tribolium castaneum is a small and low maintenance beetle that has emerged as a most suitable insect model for studying developmental biology and functional genetic analysis. Diverse population genetic studies have been conducted using Tribolium as the principal model to establish basic facts and principles of inbreeding experiments and response to the selection and other quantitative genetics fundamentals. The advanced molecular genetic studies presently focused on the use of Tribolium as a typical invertebrate model for higher diploid eukaryotes. After a whole genome sequencing of Tribolium, many areas of functional genomics were unraveled, which enabled the use of it in many technical approaches of genomics. The present text reviews the use of Tribolium in techniques such as RNAi, transgenic studies, immune priming, immunohistochemistry, in situ hybridization, gene sequencing for characterization of microRNAs, and gene editing using engineered endonuclease. In contrast to Drosophila, the T. castaneum holds a robust systemic RNAi response, which makes it an excellent model for comparative functional genetic studies. Keywords: functional genomics, hox gene, insertional mutagenesis, RNAi, Tribolium.

scholarly journals Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

Biology Open ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. bio055343 ◽  
Author(s):  
Daniel Chu ◽  
An Nguyen ◽  
Spenser S. Smith ◽  
Zuzana Vavrušová ◽  
Richard A. Schneider
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Inducible Promoter ◽  
In Ovo ◽  
Control Of Gene Expression ◽  
Promoter Construct ◽  
Avian Development ◽  
Spatiotemporal Control ◽  
Promoter System

ABSTRACTPrecisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

scholarly journals Molecular characterization of antibodies bearing Id-460. II. Molecular basis for Id-460 expression.

1985 ◽  
Vol 162 (5) ◽  
pp. 1494-1511 ◽  
Author(s):  
E A Dzierzak ◽  
P Brodeur ◽  
T Marion ◽  
C A Janeway ◽  
A Bothwell
Keyword(s):  
Molecular Basis ◽  
Molecular Genetic ◽  
Gene Segment ◽  
Binding Specificity ◽  
Antigen Binding ◽  
Measurable Effect ◽  
Variable Regions ◽  
Genetic Level

Id-460+ immunoglobulins can be induced in vivo by immunization with dinitrophenyl (DNP) or P. pneumotropica and form two nonoverlapping groups of antibodies with respect to antigen binding specificity. In this study, using Id-460+ antibodies of differing antigen binding specificities, we compared on the molecular genetic level the five gene segment combinations (VH, DH, JH, VL, and JL) that encode the variable regions of these idiotype-positive immunoglobulins. The Id-460 determinant appears to be a conformational or combinatorial determinant encoded by VH460 and VK1 crosshybridizing genes. DH, JH, and JK gene segments appear to have no measurable effect upon expression of Id-460. Finally, antigen binding specificity does not appear to simply localize to any particular gene segment but may in part be the result of somatic mutation and/or VDJH junctional sequences, whose length correlates roughly with antigen binding specificity.

DEFECTIVE STEROIDAL SYNTHESIS BY ADRENAL TISSUE: THE CONTROL OF GENE EXPRESSION CAUSING LOSS OF ACTIVE STEROIDOGENIC ENZYMES

1974 ◽  
Vol 77 (2) ◽  
pp. 325-336
Author(s):  
K. Williams
Keyword(s):  
Gene Expression ◽  
Adrenal Glands ◽  
Hydroxysteroid Dehydrogenase ◽  
Ascites Cell ◽  
Control Of Gene Expression ◽  
Adrenocortical Hyperplasia ◽  
Normal Human ◽  
Adrenocortical Tumour

ABSTRACT RNA was isolated from normal human adrenal glands and found to cause the formation of Δ5-3β-hydroxysteroid dehydrogenase-isomerase and steroid 21-hydroxylase activities by a Krebs II ascites cell-free protein synthesising system. Although no functional steroid 21-hydroxylase in vivo or in vitro was found in a gland from a patient with virilism due to congenital adrenocortical hyperplasia the RNA would still give steroid 21-hydroxylase-like activity in the protein synthesising system which suggests that the inherited defect was not in the structural gene. Activity could not be induced by RNA from a 'non-functioning' adrenocortical tumour or rat liver.

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