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In JoVE (1)
- Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy
Other Publications (6)
Articles by Ya-Fang Chiu in JoVE
Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy
Kathryn Norby1, Ya-Fang Chiu1, Bill Sugden1
1Department of Oncology, University of Wisconsin - Madison
A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.
Published December 13, 2012. Keywords: Genetics, Molecular Biology, Cellular Biology, Genomics, DNA synthesis, DNA partitioning, plasmids, lac operator, lac repressor, mammalian cells, fluorescence microscopy
Other articles by Ya-Fang Chiu on PubMed
Biochemical and Biophysical Research Communications. Feb, 2003 | Pubmed ID: 12589821
(-)-Epigallocatechin gallate (EGCG), abundant in green tea, is a potent anti-microbial and anti-tumor compound. This investigation used immunoblot, flow cytometry, microarray, and indirect immunofluorescence analyses to show that at concentrations exceeding 50 microM, EGCG inhibits the expression of Epstein-Barr virus (EBV) lytic proteins, including Rta, Zta, and EA-D, but does not affect the expression of EBNA-1. Moreover, DNA microarray and transient transfection analyses demonstrated that EGCG blocks EBV lytic cycle by inhibiting the transcription of immediate-early genes, thus inhibiting the initiation of EBV lytic cascade.
The Journal of General Virology. Sep, 2007 | Pubmed ID: 17698655
A mutant library of 249 mutants with mutations that span the entire Epstein-Barr virus (EBV) genome was generated by transposition with EZ : : TN
The Journal of General Virology. Oct, 2008 | Pubmed ID: 18796711
BRCA1-associated protein 2 (BRAP2) is known to interact with the kinase suppressor of Ras 1 (KSR1), inhibiting the ERK signal transduction cascade. This study found that an Epstein-Barr virus (EBV) immediate-early protein, Rta, is a binding partner of BRAP2 in yeast and confirmed the binding in vitro by a glutathione S-transferase pull-down assay and in vivo by co-immunoprecipitation in 293(maxi-EBV) cells. Binding studies also showed that Rta and KSR1 interacted with the C-terminal 202 aa region in BRAP2. Additionally, Rta appeared to prevent the binding of KSR1 to BRAP2, activating the ERK signal transduction pathway and the transcription of an EBV immediate-early gene, BZLF1. Activation of the ERK signal transduction pathway by Rta may be critical for the maintenance of the lytic state of EBV.
Molecules (Basel, Switzerland). 2010 | Pubmed ID: 20948499
Reactivation of Epstein-Barr virus (EBV) from latency to the lytic cycle is required for the production of viral particles. Here, we examine the capacity of resveratrol to inhibit the EBV lytic cycle. Our results show that resveratrol inhibits the transcription of EBV immediate early genes, the expression of EBV lytic proteins, including Rta, Zta, and EA-D and reduces viron production, suggesting that this compound may be useful for preventing the proliferation of the virus.
Characterization and Intracellular Trafficking of Epstein-Barr Virus BBLF1, a Protein Involved in Virion Maturation
Journal of Virology. Sep, 2012 | Pubmed ID: 22740416
Epstein-Barr virus (EBV) BBLF1 shares 13 to 15% amino acid sequence identities with the herpes simplex virus 1 UL11 and cytomegalovirus UL99 tegument proteins, which are involved in the final envelopment during viral maturation. This study demonstrates that BBLF1 is a myristoylated and palmitoylated protein, as are UL11 and UL99. Myristoylation of BBLF1 both facilitates its membrane anchoring and stabilizes it. BBLF1 is shown to localize to the trans-Golgi network (TGN) along with gp350/220, a site where final envelopment of EBV particles takes place. The localization of BBLF1 at the TGN requires myristoylation and two acidic clusters, which interact with PACS-1, a cytosolic protein, to mediate retrograde transport from the endosomes to the TGN. Knockdown of the expression of BBLF1 during EBV lytic replication reduces the production of virus particles, demonstrating the requirement of BBLF1 to achieve optimal production of virus particles. BBLF1 is hypothesized to facilitate the budding of tegumented capsid into glycoprotein-embedded membrane during viral maturation.
PloS One. 2012 | Pubmed ID: 23091622
Epstein-Barr virus is a gamma-herpes virus that is causally associated with several lymphomas and carcinomas. This virus encodes at least 25 pre-miRNAs, which are expressed in infected cells to yield more than 50 detected mature miRNAs. miRNAs are small, non-coding RNAs that inhibit gene expression by promoting the inhibition of translation or of degradation of mRNAs. Currently, the function of these viral miRNAs and the contribution they provide to EBV's life-cycle remain largely unknown, due to difficulties in identifying cellular and viral genes regulated by these miRNAs. We have compared and contrasted two methods to identify targets of viral miRNAs in order to identify the advantages and limitations of each method to aid in uncovering the functions of EBV's miRNAs.