scholarly journals Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha

2020 ◽  
Author(s):  
Susanna Sauret-Güeto ◽  
Eftychios Frangedakis ◽  
Linda Silvestri ◽  
Marius Rebmann ◽  
Marta Tomaselli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chloroplast Transformation ◽  
Cellular Growth ◽  
Marchantia Polymorpha ◽  
Promoter Sequences ◽  
Vector Systems ◽  
Gene Redundancy ◽  
Dna Elements ◽  
Living Tissues

ABSTRACTWe present the OpenPlant toolkit, a set of interlinked resources and techniques to develop Marchantia as testbed for bioengineering in plants. Marchantia is a liverwort, a simple plant with an open form of development that allows direct visualization of gene expression and dynamics of cellular growth in living tissues. We describe new techniques for simple and efficient axenic propagation and maintenance of Marchantia lines with no requirement for glasshouse facilities. Marchantia plants spontaneously produce clonal propagules within a few weeks of regeneration, and lines can be amplified million-fold in a single generation by induction of the sexual phase of growth, crossing and harvesting of progeny spores. The plant has a simple morphology and genome with reduced gene redundancy, and the dominant phase of its life cycle is haploid, making genetic analysis easier. We have built robust Loop assembly vector systems for nuclear and chloroplast transformation and genome editing. These have provided the basis for building and testing a modular library of standardized DNA elements with highly desirable properties. We have screened transcriptomic data to identify a range of candidate genes, extracted putative promoter sequences, and tested them in vivo to identify new constitutive promoter elements. The resources have been combined into a toolkit for plant bioengineering that is accessible for laboratories without access to traditional facilities for plant biology research. The toolkit is being made available under the terms of the OpenMTA and will facilitate the establishment of common standards and the use of this simple plant as testbed for synthetic biology.

Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice

1992 ◽  
Vol 12 (9) ◽  
pp. 3978-3990
Author(s):  
B Liu ◽  
G D Hammer ◽  
M Rubinstein ◽  
M Mortrud ◽  
M J Low
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Intermediate Lobe ◽  
Mobility Shift ◽  
Specific Expression ◽  
Dna Elements ◽  
Pomc Gene

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

scholarly journals Versatile Retrovirus Vector Systems for Regulated Gene Expression In Vitro and In Vivo

1997 ◽  
Vol 3 (7) ◽  
pp. 466-476 ◽  
Author(s):  
Dirk Lindemann ◽  
Edward Patriquin ◽  
Sandy Feng ◽  
Richard C. Mulligan
Keyword(s):  
Gene Expression ◽  
Vector Systems ◽  
Retrovirus Vector ◽  
Regulated Gene Expression

scholarly journals Tat has a dual role in simian immunodeficiency virus transcription

2012 ◽  
Vol 93 (10) ◽  
pp. 2279-2289 ◽  
Author(s):  
Gisela J. van der Velden ◽  
Monique A. Vink ◽  
Ben Berkhout ◽  
Atze T. Das
Keyword(s):  
Gene Expression ◽  
Simian Immunodeficiency Virus ◽  
Cultured Cells ◽  
Virus Transcription ◽  
Immunodeficiency Virus ◽  
Dna Elements ◽  
Tar Rna ◽  
Essential Function ◽  
Expression Mechanism

Tat has a pivotal role in human and simian immunodeficiency virus (HIV and SIV) replication because it stimulates transcription by binding to the trans-activator response (TAR) element. In addition, several other Tat functions have been proposed. Most studies have focused on HIV-1 Tat and much less is known about SIV Tat. An SIVmac239 variant was constructed previously in which the Tat–TAR transcription mechanism is functionally replaced by the doxycycline-inducible Tet-On gene expression mechanism (SIV-rtTA). In this study, SIV-rtTA variants were used to analyse the functions of SIV Tat. It was shown that Tat-minus SIV-rtTA variants replicated efficiently in PM1 T-cells, ruling out an additional essential Tat function. Nevertheless, replication was suboptimal in other cells, and evolutionary pressure to repair Tat expression was documented. It was demonstrated that SIV-rtTA required Tat for optimal gene expression, despite the absence of the Tat–TAR axis. This Tat effect was lost upon replacement of the long terminal repeat promoter region by a non-related promoter. These results indicate that Tat can activate SIV transcription via TAR RNA and U3 DNA elements but has no other essential function in replication in cultured cells. The experiments were limited to cell lines and PBMCs, and did not exclude an accessory Tat function under specific conditions or in vivo.

scholarly journals Control of differentiation-induced calbindin-D9k gene expression in Caco-2 cells by cdx-2 and HNF-1α

2004 ◽  
Vol 287 (5) ◽  
pp. G943-G953 ◽  
Author(s):  
Liyong Wang ◽  
Anna Klopot ◽  
Jean-Noel Freund ◽  
Lauren N. Dowling ◽  
Stephen D. Krasinski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Calcium Absorption ◽  
Hepatocyte Nuclear Factor ◽  
Promoter Sequences ◽  
Physiological Importance ◽  
Calbindin D9k ◽  
Enhancer Binding Protein ◽  
Promoter Construct ◽  
Calbindin D

Calbindin D9k (CaBP) is critical for intestinal calcium absorption; its in vivo expression is restricted to differentiated enterocytes of the small intestine. Our goal was to identify factors controlling the transcriptional regulation of this gene in the human intestine. Both the natural gene and a 4600-bp promoter construct were strongly regulated by differentiation (>100-fold) but not by treatment with 1,25(OH)2 vitamin D (<2-fold) in the Caco-2 clone TC7. Deletion-mutation studies revealed that conserved promoter sequences for cdx-2 (at −3158 bp) and hepatocyte nuclear factor (HNF)-1 (at −3131 and at −98 bp) combined to control CaBP expression during differentiation. Other putative response elements were not important for CaBP regulation in TC7 cells (CCAAT enhancer binding protein, pancreatic duodenal homebox-1 (pdx-1), a proximal cdx-2 element). Mutation of the distal HNF-1 site had the greatest impact on CaBP gene expression through disruption of HNF-1α binding; both basal and differentiation-mediated CaBP expression was reduced by 80%. In contrast, mutation of the distal cdx-2 element reduced only basal CaBP expression. Whereas a 60% reduction of CaBP mRNA in the duodenum of HNF-1α null mice confirmed the physiological importance of HNF-1α for CaBP gene regulation, additional studies showed that maximal CaBP expression requires the presence of both HNF-1α and cdx-2. Our data suggest that cdx-2 is a permissive factor that influences basal CaBP expression in enterocytes and that HNF-1α modulates CaBP gene expression during cellular differentiation.

scholarly journals Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice.

1992 ◽  
Vol 12 (9) ◽  
pp. 3978-3990 ◽  
Author(s):  
B Liu ◽  
G D Hammer ◽  
M Rubinstein ◽  
M Mortrud ◽  
M J Low
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Intermediate Lobe ◽  
Mobility Shift ◽  
Specific Expression ◽  
Dna Elements ◽  
Pomc Gene

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

scholarly journals Normative models of enhancer function

2020 ◽  
Author(s):  
Rok Grah ◽  
Benjamin Zoller ◽  
Gašper Tkačik
Keyword(s):  
Gene Expression ◽  
High Specificity ◽  
Equilibrium Models ◽  
Gene Expression Noise ◽  
Promoter Sequences ◽  
Physical Measurements ◽  
Enhancer Function ◽  
Non Equilibrium

In prokaryotes, thermodynamic models of gene regulation provide a highly quantitative mapping from promoter sequences to gene expression levels that is compatible with in vivo and in vitro bio-physical measurements. Such concordance has not been achieved for models of enhancer function in eukaryotes. In equilibrium models, it is difficult to reconcile the reported short transcription factor (TF) residence times on the DNA with the high specificity of regulation. In non-equilibrium models, progress is difficult due to an explosion in the number of parameters. Here, we navigate this complexity by looking for minimal non-equilibrium enhancer models that yield desired regulatory phenotypes: low TF residence time, high specificity and tunable cooperativity. We find that a single extra parameter, interpretable as the “linking rate” by which bound TFs interact with Mediator components, enables our models to escape equilibrium bounds and access optimal regulatory phenotypes, while remaining consistent with the reported phenomenology and simple enough to be inferred from upcoming experiments. We further find that high specificity in non-equilibrium models is in a tradeoff with gene expression noise, predicting bursty dynamics — an experimentally-observed hallmark of eukaryotic transcription. By drastically reducing the vast parameter space to a much smaller subspace that optimally realizes biological function prior to inference from data, our normative approach holds promise for mathematical models in systems biology.

scholarly journals A toolkit to generate inducible and interconvertible Drosophila transgenes

2020 ◽  
Author(s):  
Franz Wendler ◽  
Sangbin Park ◽  
Claire Hill ◽  
Alessia Galasso ◽  
Kathleen R. Chang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binary Systems ◽  
Expression System ◽  
Fruit Fly ◽  
Regulatory Elements ◽  
Conditional Gene Expression ◽  
Dna Elements ◽  
Independent Site ◽  
Inducible Gene

ABSTRACTThe existence of three independent binary systems for conditional gene expression (Gal4/UAS; LexA/LexAop; QF/QUAST) has greatly expanded versatile genetic analyses in the fruit fly Drosophila melanogaster; however, the experimental application of these tools is limited by the need to generate multiple collections of non-interchangeable transgenic fly strains for each inducible gene expression system. To address this practical limitation, we developed a multipurpose modular vector that contains the regulatory elements from all three binary systems, enabling Gal4-, LexA- or QF-dependent expression of transgenes. Our methods also incorporate DNA elements that facilitate independent site-specific recombination and elimination of regulatory UAS, LexAop or QUAST modules with spatial and temporal control, thus offering unprecedented possibilities and logistical advantages for in vivo genetic modulation and efficient interconversion of transgenic fly lines.

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