Spontaneous reactivation of a site-specifically placed transgene independent of copy number or DNA methylation

2019 ◽  
Vol 71 (4) ◽  
pp. 1574-1584
Author(s):  
Junjie Wei ◽  
Zhicheng Dong ◽  
David W Ow
Keyword(s):  
Chromatin Modification ◽  
Scale Production ◽  
Dna Hypermethylation ◽  
Gfp Expression ◽  
Long Term Stability ◽  
Gfp Reporter ◽  
Gene Reactivation ◽  
Gfp Reporter Gene ◽  
A Site

Abstract As millions of seeds are produced from a breeding line, the long-term stability of transgene expression is vital for commercial-scale production of seeds with transgenic traits. Transgenes can be silenced by epigenetic mechanisms, but reactivation of expression can occur as a result of treatment with chromatin modification inhibitors such as 5-azacytidine, from stress such as heat or UV-B, or in mutants that have acquired a defect in gene silencing. Previously, we targeted a gfp reporter gene into the tobacco (Nicotiana tabacum) genome by site-specific recombination but still found some silenced lines among independent integration events. One such line also had a second random copy and both copies showed DNA hypermethylation. To test whether removing the second copy would reactivate gfp expression, two T1 plants were backcrossed to the wild type. Whereas the silenced status was maintained in the progenies from one backcross, spontaneous partial reactivation of gfp expression was found among progenies from a second backcross. However, this reactivation did not correlate with loss of the second random copy or with a significant change in the pattern or amount of DNA hypermethylation. This finding supports the suggestion that gene reactivation does not necessarily involve loss of DNA homology or methylation.

scholarly journals Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

1999 ◽  
Vol 67 (4) ◽  
pp. 1812-1820 ◽  
Author(s):  
Maurizio del Poeta ◽  
Dena L. Toffaletti ◽  
Thomas H. Rude ◽  
Sara D. Sparks ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Cns Infection ◽  
Gfp Expression ◽  
Gfp Reporter ◽  
Gfp Reporter Gene ◽  
Differential Gene ◽  
Green Fluorescent

ABSTRACT Synthetic green fluorescent protein (GFP) was used as a reporter to detect differential gene expression in the pathogenic fungusCryptococcus neoformans. Promoters from the C. neoformans actin, GAL7, or mating-type alpha pheromone (MFα1) genes were fused to GFP, and the resulting reporter genes were used to assess gene expression in serotype A C. neoformans. Yeast cells containing an integrated pACT::GFP construct demonstrated that the actin promoter was expressed during vegetative growth on yeast extract-peptone-dextrose medium. In contrast, yeast cells containing the inducible GAL7::GFP or MFα1::GFP reporter genes expressed significant GFP activity only during growth on galactose medium or V-8 agar, respectively. These findings demonstrated that the GAL7 and MFα1 promoters from a serotype D C. neoformans strain function when introduced into a serotype A strain. Because the MFα1 promoter is induced by nutrient deprivation and the MATα locus containing the MFα1 gene has been linked with virulence, yeast cells containing the pMFα1::GFP reporter gene were analyzed for GFP expression in the central nervous system (CNS) of immunosuppressed rabbits. In fact, significant GFP expression from the MFα1::GFP reporter gene was detected after the first week of a CNS infection. These findings suggest that there are temporal, host-specific cues that regulate gene expression during infection and that the MFα1 gene is induced during the proliferative stage of a CNS infection. In conclusion, GFP can be used as an effective and sensitive reporter to monitor specific C. neoformans gene expression in vitro, and GFP reporter constructs can be used as an approach to identify a novel gene(s) or to characterize known genes whose expression is regulated during infection.

Rehabilitation of field tunnel erosion using techniques developed for construction with dispersive soils

Soil Research ◽  
2007 ◽  
Vol 45 (4) ◽  
pp. 280 ◽  
Author(s):  
M. A. Hardie ◽  
W. E. Cotching ◽  
P. R. Zund
Keyword(s):  
Optical Fibre ◽  
Soil Analysis ◽  
Long Term Stability ◽  
Site Investigations ◽  
Construction Techniques ◽  
A Site ◽  
Dispersive Soils ◽  
Tunnel System ◽  
Surface And Subsurface Flow

Past repairs of field tunnel erosion using mechanical treatments (deep ripping, contour furrowing, contour ripping, etc.) and reestablishment of perennial vegetation have often failed, resulting in further tunnelling. Techniques to prevent ‘piping’ (tunnel erosion) in earth dams constructed using sodic clays have not been used in the repair of field tunnel erosion; however, these techniques have the potential to reduce recurrent failure. Installation of an optical fibre cable in a Grey Sodosol and Grey Dermosol at a site near Dunalley, Tasmania, in November 2001, resulted in the formation of a 380-m-long tunnel erosion system. Detailed site investigations and soil analysis in 2004 indicated that the tunnel erosion resulted from the consequences of inadequate compaction during the installation of the optical fibre cable, and secondary processes such as capture of surface and subsurface flow, dispersion of sodic clays, and translocation of dispersed clay platelets through the poorly compacted fill. Repair works consisted of excavating the entire length of the tunnel system, chemical amelioration with gypsum, compaction of repacked fill to reduce internal porosity, and installation of sand blocks to capture and remove water moving along the reinstalled cable. Inspections conducted 2 years after completion of the repair works indicated the site to be stable; however, it is acknowledged that this is too short an interval to adequately assess the long-term stability of repair works and techniques employed at the site. Due to the costs associated with the application of dam construction techniques, their use for the repair of field tunnel erosion is likely to only be justified where continued erosion poses a risk of damage to high value infrastructure.

scholarly journals High-Frequency Epigenetic Repression and Silencing of Retroviruses Can Be Antagonized by Histone Deacetylase Inhibitors and Transcriptional Activators, but Uniform Reactivation in Cell Clones Is Restricted by Additional Mechanisms

2007 ◽  
Vol 81 (6) ◽  
pp. 2592-2604 ◽  
Author(s):  
Richard A. Katz ◽  
Emily Jack-Scott ◽  
Anna Narezkina ◽  
Ivan Palagin ◽  
Pamela Boimel ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Histone Deacetylase ◽  
High Frequency ◽  
Reporter Expression ◽  
Gfp Expression ◽  
Epigenetic Effects ◽  
Cell Clones ◽  
Gfp Reporter ◽  
Gfp Reporter Gene ◽  
Epigenetic Repression

ABSTRACT Integrated retroviral DNA is subject to epigenetic gene silencing, but the viral and host cell properties that influence initiation, maintenance, and reactivation are not fully understood. Here we describe rapid and high-frequency epigenetic repression and silencing of integrated avian sarcoma virus (ASV)-based vector DNAs in human HeLa cells. Initial studies utilized a vector carrying the strong human cytomegalovirus (hCMV) immediate-early (IE) promoter to drive expression of a green fluorescent protein (GFP) reporter gene, and cells were sorted into two populations based on GFP expression [GFP(+) and GFP(−)]. Two potent epigenetic effects were observed: (i) a very broad distribution of GFP intensities among cells in the GFP(+) population as well as individual GFP(+) clones and (ii) high-frequency GFP reporter gene silencing in GFP(−) cells. We previously showed that histone deacetylases (HDACs) can associate with ASV DNA soon after infection and may act to repress viral transcription at the level of chromatin. Consistent with this finding, we report here that treatment with the histone deacetylase inhibitor trichostatin A (TSA) induces GFP activation in GFP(−) cells and can also increase GFP expression in GFP(+) cells. In the case of the GFP(−) populations, we found that after removal of TSA, GFP silencing was reestablished in a subset of cells. We used that finding to enrich for stable GFP(−) cell populations in which viral GFP reporter expression could be reactivated by TSA; furthermore, we found that the ability to isolate such populations was independent of the promoter driving the GFP gene. In such enriched cultures, hCMV IE-driven, but not the viral long terminal repeat-driven, silent GFP reporter expression could be reactivated by the transcriptional activator prostratin. Microscopy-based studies using synchronized cells revealed variegated reactivation in cell clones, indicating that secondary epigenetic effects can restrict reactivation from silencing. Furthermore we found that entry into S phase was not required for reactivation. We conclude that HDACs can act rapidly to initiate and maintain promoter-independent retroviral epigenetic repression and silencing but that reactivation can be restricted by additional mechanisms.

scholarly journals PRIORITY EFFECTS AND ECOLOGICAL RESTORATION

2020 ◽  
Author(s):  
Emanuela W. A. Weidlich ◽  
Cara R Nelson ◽  
John L Maron ◽  
Ragan M. Callaway4 ◽  
Benjamin M. Delory ◽  
...  
Keyword(s):  
Ecological Restoration ◽  
Priority Effects ◽  
Future Research ◽  
Temperate Grasslands ◽  
Long Term Stability ◽  
Lack Of Information ◽  
General Importance ◽  
Restoration Practice ◽  
A Site

Priority effects refer to the order or timing of species arrival, including how species that arrive early to a site either positively or negatively affect establishment, growth, or reproduction of species that arrive later. Despite clear implications of priority effects on ecological restoration, to date there are no reviews of how and where priority effects have been studied and the extent to findings can be applied to restoration practice. Here, we survey the literature on priority effects and a) summarize patterns that are relevant to restoration; b) synthesize information on the mechanisms through which priority effects operate, and on how these mechanisms can be manipulated to achieve particular restoration goals; and c) highlight potential future research needed to improve use of priority effects in restoration. We found that even small delays in arrival time, as opposed to simultaneous arrival of species, can promote differences in subsequent community composition. Even so, there have been very few studies on the long-term stability of these priority effects, and the majority were conducted in temperate grasslands. Given the lack of information for other biomes, the general importance of priority effects, as well as its application to restoration, is unknown. Our findings suggest that creating alternative vegetation states via priority treatments might be a promising avenue to further explore, but that for the concept to be operationalized for restoration practice there is a need for research in the diverse types of ecosystems that are priorities for restoration and that occurs over longer time periods.

Stability and Change in Emotional Intelligence: Exploring the Transition to Young Adulthood

2005 ◽  
Vol 26 (2) ◽  
pp. 100-106 ◽  
Author(s):  
James D.A. Parker ◽  
Donald H. Saklofske ◽  
Laura M. Wood ◽  
Jennifer M. Eastabrook ◽  
Robyn N. Taylor
Keyword(s):  
High School ◽  
Emotional Intelligence ◽  
Postsecondary Education ◽  
Life Transition ◽  
Full Time ◽  
Major Life ◽  
Long Term Stability ◽  
Time Period ◽  
Transition From High School

Abstract. The concept of emotional intelligence (EI) has attracted growing interest from researchers working in various fields. The present study examined the long-term stability (32 months) of EI-related abilities over the course of a major life transition (the transition from high school to university). During the first week of full-time study, a large group of undergraduates completed the EQ-i:Short; 32 months later a random subset of these students (N = 238), who had started their postsecondary education within 24 months of graduating from high school, completed the measures for a second time. The study found EI scores to be relatively stable over the 32-month time period. EI scores were also found to be significantly higher at Time 2; the overall pattern of change in EI-levels was more than can be attributed to the increased age of the participants.

Multi-Center Calibration of the Second Reference Material for Thromboplastin, Rabbit, Plain, Coded CRM 149R

1991 ◽  
Vol 65 (03) ◽  
pp. 263-267 ◽  
Author(s):  
A M H P van den Besselaar ◽  
R M Bertina
Keyword(s):  
Reference Material ◽  
Standard Error ◽  
Standard Errors ◽  
Sensitivity Index ◽  
Long Term Stability ◽  
Significant Bias ◽  
The Mean ◽  
The Relationship ◽  
International Sensitivity Index

SummaryIn a collaborative trial of eleven laboratories which was performed mainly within the framework of the European Community Bureau of Reference (BCR), a second reference material for thromboplastin, rabbit, plain, was calibrated against its predecessor RBT/79. This second reference material (coded CRM 149R) has a mean International Sensitivity Index (ISI) of 1.343 with a standard error of the mean of 0.035. The standard error of the ISI was determined by combination of the standard errors of the ISI of RBT/79 and the slope of the calibration line in this trial.The BCR reference material for thromboplastin, human, plain (coded BCT/099) was also included in this trial for assessment of the long-term stability of the relationship with RBT/79. The results indicated that this relationship has not changed over a period of 8 years. The interlaboratory variation of the slope of the relationship between CRM 149R and RBT/79 was significantly lower than the variation of the slope of the relationship between BCT/099 and RBT/79. In addition to the manual technique, a semi-automatic coagulometer according to Schnitger & Gross was used to determine prothrombin times with CRM 149R. The mean ISI of CRM 149R was not affected by replacement of the manual technique by this particular coagulometer.Two lyophilized plasmas were included in this trial. The mean slope of relationship between RBT/79 and CRM 149R based on the two lyophilized plasmas was the same as the corresponding slope based on fresh plasmas. Tlowever, the mean slope of relationship between RBT/79 and BCT/099 based on the two lyophilized plasmas was 4.9% higher than the mean slope based on fresh plasmas. Thus, the use of these lyophilized plasmas induced a small but significant bias in the slope of relationship between these thromboplastins of different species.

The Stability of the WHO Reference Thromboplastin NIBS & C 67/40

1979 ◽  
Vol 42 (04) ◽  
pp. 1135-1140 ◽  
Author(s):  
G I C Ingram
Keyword(s):  
Human Brain ◽  
Collaborative Study ◽  
Calibration Constant ◽  
Long Term Stability ◽  
Freeze Dried ◽  
Show Evidence ◽  
Human Material ◽  
Reference Preparation ◽  
The Stability

SummaryThe International Reference Preparation of human brain thromboplastin coded 67/40 has been thought to show evidence of instability. The evidence is discussed and is not thought to be strong; but it is suggested that it would be wise to replace 67/40 with a new preparation of human brain, both for this reason and because 67/40 is in a form (like Thrombotest) in which few workers seem to use human brain. A �plain� preparation would be more appropriate; and a freeze-dried sample of BCT is recommended as the successor preparation. The opportunity should be taken also to replace the corresponding ox and rabbit preparations. In the collaborative study which would be required it would then be desirable to test in parallel the three old and the three new preparations. The relative sensitivities of the old preparations could be compared with those found in earlier studies to obtain further evidence on the stability of 67/40; if stability were confirmed, the new preparations should be calibrated against it, but if not, the new human material should receive a calibration constant of 1.0 and the new ox and rabbit materials calibrated against that.The types of evidence available for monitoring the long-term stability of a thromboplastin are discussed.

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