transfection experiment
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Epigenomics ◽  
2019 ◽  
Vol 11 (14) ◽  
pp. 1581-1599 ◽  
Author(s):  
Elisa Reale ◽  
Daniela Taverna ◽  
Laura Cantini ◽  
Loredana Martignetti ◽  
Matteo Osella ◽  
...  

Aim: Growing evidence shows a strong interplay between post-transcriptional regulation, mediated by miRNAs (miRs) and epigenetic regulation. Nevertheless, the number of experimentally validated miRs (called epi-miRs) involved in these regulatory circuitries is still very small. Material & methods: We propose a pipeline to prioritize candidate epi-miRs and to identify potential epigenetic interactors of any given miR starting from miR transfection experiment datasets. Results & conclusion: We identified 34 candidate epi-miRs: 19 of them are known epi-miRs, while 15 are new. Moreover, using an in-house generated gene expression dataset, we experimentally proved that a component of the polycomb-repressive complex 2, the histone methyltransferase enhancer of zeste homolog 2 (EZH2), interacts with miR-214, a well-known prometastatic miR in melanoma and breast cancer, highlighting a miR-214-EZH2 regulatory axis potentially relevant in tumor progression.


2019 ◽  
Vol 46 (6) ◽  
pp. 6371-6379
Author(s):  
Victoria Drechsel ◽  
Birgit Fiechtner ◽  
Martina Höckner

Abstract The regulation of metallothionein (MT) gene expression as important part of the detoxification machinery is only scarcely known in invertebrates. In vertebrates, MT gene activation is mediated by the metal-transcription factor 1 (MTF-1) binding to metal response elements (MREs). In invertebrates, the mechanisms of MT gene activation seems to be more diverse. In some invertebrate species, MTF-1 orthologues as well as their ability to activate MT genes via MREs have been uncovered. Although earthworm MTs have been well studied, a MTF-1 orthologue has not yet been described and MT gene activation mechanisms are largely unknown. Analyses of the earthworm wMT2 promoter by reporter gene assays have been performed. We could show that the wMT2 promoter was active in mouse embryonic fibroblasts (NIH/3T3) as well as in mouse MTF-1−/−cells (DKO7). The presence of mouse MTF-1 (mMTF1) led to a significant increase in reporter gene activity. We observed that cadmium as well as zinc had an effect on promoter activity. In the presence of zinc, promoter activity doubled in NIH cells, however, we did not observe a significant effect in the DKO7 cell line. Cadmium decreased promoter activity in DKO7 cells, but this effect could be reversed by providing mMTF1 in a co-transfection experiment. We suggest that MT gene expression in the earthworm is not entirely dependent on a MRE binding protein. Interestingly, the shortest promoter fragment including MRE1 showed the highest promoter activity under control conditions.


2011 ◽  
Vol 177 (2) ◽  
pp. 156-159 ◽  
Author(s):  
Voravuth Somsak ◽  
Somdet Srichairatanakool ◽  
Sumalee Kamchonwongpaisan ◽  
Yongyuth Yuthavong ◽  
Chairat Uthaipibull

2004 ◽  
Vol 287 (2) ◽  
pp. G352-G362 ◽  
Author(s):  
Nary Veal ◽  
Chih-Lin Hsieh ◽  
Shigang Xiong ◽  
Jose M. Mato ◽  
Shelly Lu ◽  
...  

S-adenosylmethionine (SAM) is the principal biological methyl donor and precursor for polyamines. SAM is known to be hepatoprotective in many liver disease models in which TNF-α is implicated. The present study investigated whether and how SAM inhibited LPS-stimulated TNF-α expression in Kupffer cells (hepatic macrophages). SAM downregulated TNF-α expression in LPS-stimulated Kupffer cells at the transcriptional level as suggested by a transfection experiment with a TNF-α promoter-reporter gene. This inhibition was not mediated through decreased NF-κB binding to four putative κB binding elements located within the promoter. The inhibited promoter activity was neither prevented by overexpression of p65 and/or its coactivator p300 nor enhanced by overexpression of coactivator-associated arginine methyltransferase-1, an enzyme that methylates p300 and inhibits a p65-p300 interaction. SAM did not lead to DNA methylation at the most common CpG target sites in the TNF-α promoter. Moreover, 5′-methylthioadenosine (MTA), which is derived from SAM but does not serve as a methyl donor, recapitulated SAM's effect with more potency. These data demonstrate that SAM inhibits TNF-α expression at the level downstream of NF-κB binding and at the level of the promoter activity via mechanisms that do not appear to involve the limited availability of p65 or p300. Furthermore, our study is the first to demonstrate a potent inhibitory effect on NF-κB promoter activity and TNF-α expression by a SAM's metabolite, MTA.


1996 ◽  
Vol 39 (1-2) ◽  
pp. 68-78 ◽  
Author(s):  
Maxime Chireux ◽  
Estelle Espinos ◽  
Stéphane Bloch ◽  
Minoru Yoshida ◽  
Michel J. Weber

1988 ◽  
Vol 8 (2) ◽  
pp. 938-944
Author(s):  
H Uemura ◽  
R B Wickner

For the maintenance of "killer" M1 double-stranded RNA in Saccharomyces cerevisiae, more than 30 chromosomal genes are required. The requirement for some of these genes can be completely suppressed by a cytoplasmic element, [B] (for bypass). We have isolated a mutant unable to maintain [B] (mab) and found that it is allelic to MAK10, one of the three chromosomal MAK genes required for the maintenance of L-A. The heat curing of [B] always coincided with the loss of L-A. To confirm that [B] is located on L-A, we purified viral particles containing either L-A or M1 from strains with or without [B] activity and transfected these purified particles into a strain which did not have either L-A or M1. The transfectants harboring L-A and M1 from a [B] strain showed the [B] phenotype, but the transfectants with L-A and M1 from a [B-o] strain did not show the [B] phenotype. Furthermore, the transfectants having L-A from a [B] strain and M1 from a [B-o] strain also showed the [B] phenotype. Therefore, we concluded that [B] is a property of a variant of L-A. In the transfection experiment, we also proved that the superkiller phenotype of the [B] strain is a property of L-A and that L-A with [B] activity can maintain a higher copy number of M1 regardless of the source of M1 viruslike particles. These data suggest that MAK genes whose mutations are suppressed by [B] are concerned with the protection of M1 (+) single-stranded RNA or the formation of M1 viruslike particles and that an L-A with more efficient production of M1 viruslike particles can completely dispense with the requirement for those MAK genes.


1988 ◽  
Vol 8 (2) ◽  
pp. 938-944 ◽  
Author(s):  
H Uemura ◽  
R B Wickner

For the maintenance of "killer" M1 double-stranded RNA in Saccharomyces cerevisiae, more than 30 chromosomal genes are required. The requirement for some of these genes can be completely suppressed by a cytoplasmic element, [B] (for bypass). We have isolated a mutant unable to maintain [B] (mab) and found that it is allelic to MAK10, one of the three chromosomal MAK genes required for the maintenance of L-A. The heat curing of [B] always coincided with the loss of L-A. To confirm that [B] is located on L-A, we purified viral particles containing either L-A or M1 from strains with or without [B] activity and transfected these purified particles into a strain which did not have either L-A or M1. The transfectants harboring L-A and M1 from a [B] strain showed the [B] phenotype, but the transfectants with L-A and M1 from a [B-o] strain did not show the [B] phenotype. Furthermore, the transfectants having L-A from a [B] strain and M1 from a [B-o] strain also showed the [B] phenotype. Therefore, we concluded that [B] is a property of a variant of L-A. In the transfection experiment, we also proved that the superkiller phenotype of the [B] strain is a property of L-A and that L-A with [B] activity can maintain a higher copy number of M1 regardless of the source of M1 viruslike particles. These data suggest that MAK genes whose mutations are suppressed by [B] are concerned with the protection of M1 (+) single-stranded RNA or the formation of M1 viruslike particles and that an L-A with more efficient production of M1 viruslike particles can completely dispense with the requirement for those MAK genes.


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