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Bach I.,Program in Gene Function and Expression | Bach I.,University of Massachusetts Medical School
Cell Research | Year: 2012

One of the two X chromosomes in cells of female mammals is transcriptionally silenced in a process known as X chromosome inactivation (XCI). Initiation of XCI is regulated by the ubiquitin ligase Rnf12/RLIM, but the mechanisms by which Rnf12/RLIM mediates this process has been a mystery. A recent study by Gontan et al. shows that Rnf12/RLIM targets REX1, an inhibitor of XCI, for proteasomal degradation, providing an answer to this question. © 2012 IBCB, SIBS, CAS All rights reserved.


Oslowski C.M.,Program in Gene Function and Expression | Urano F.,Program in Gene Function and Expression | Urano F.,University of Massachusetts Medical School
Current Opinion in Endocrinology, Diabetes and Obesity | Year: 2010

Purpose of Review: β-Cell death is an important pathogenic component of both type 1 and type 2 diabetes. However, the specific molecular pathways and interactions involved in this process are not completely understood. Increasing evidence indicates that a type of cell stress called endoplasmic reticulum stress (ER stress) plays an important role in β-cell death. In the present article, we discuss a potential paradigm of ER stress-mediated β-cell death. Recent Findings: Upon ER stress conditions, a signaling network termed the unfolded protein response (UPR) is activated. The UPR regulates adaptive effectors to attenuate ER stress and restore ER homeostasis promoting cell survival. Paradoxically the UPR also regulates apoptotic effectors. When adaptive effectors fail to attenuate ER stress, these apoptotic effectors take into effect leading to cell death. The nature of this switch between life and death is currently under study. Summary: Depending on the nature of the stress condition, the UPR either protects β cells or promotes their death. The mechanisms of this switch are not well understood but involve the balance between adaptive and apoptotic factors regulated by the UPR. In the present article, we Review examples of this UPR balancing act between life and death and the potential mechanisms involved. © 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.


Singh A.,Program in Gene Function and Expression | Nirala N.K.,Program in Gene Function and Expression | Das S.,Hamdard University | Narula A.,Hamdard University | And 2 more authors.
Acta Physiologiae Plantarum | Year: 2011

Scopolamine is widely used for its anticholinergic properties. Because of higher physiological activity and less side effects the world demand of scopolamine is estimated to be ten times greater than other anticholinergic agents, hyoscyamine and atropine. Since natural production is limited, alternatives are required to boost the production. We report the introduction of mouse odc gene of polyamine biosynthesis pathway which is also the primary pathway of tropane alkaloids in Datura innoxia. Polyamines, mainly putrescine, serve as the common metabolite for tropane alkaloids and nicotine. We have overexpressed odc gene to modulate the metabolic flux downstream and eventually achieved higher accumulation of scopolamine in transgenic plants. Among six independent transformed lines one line (O10) produced scopolamine (0. 258 μg/g dry weight) almost six times higher than that produced by control plants (0. 042 μg/g DW). To our knowledge, this is the first report of odc overexpression in D. innoxia leading to higher scopolamine yield. © 2011 Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.


Christensen R.G.,University of Washington | Gupta A.,Program in Gene Function and Expression | Gupta A.,University of Massachusetts Medical School | Zuo Z.,University of Washington | And 4 more authors.
Nucleic Acids Research | Year: 2011

We examine the use of high-throughput sequencing on binding sites recovered using a bacterial one-hybrid (B1H) system and find that improved models of transcription factor (TF) binding specificity can be obtained compared to standard methods of sequencing a small subset of the selected clones. We can obtain even more accurate binding models using a modified version of B1H selection method with constrained variation (CV-B1H). However, achieving these improved models using CV-B1H data required the development of a new method of analysis-GRaMS (Growth Rate Modeling of Specificity)-that estimates bacterial growth rates as a function of the quality of the recognition sequence. We benchmark these different methods of motif discovery using Zif268, a well-characterized C2H2 zinc-finger TF on both a 28bp randomized library for the standard B1H method and on 6bp randomized library for the CV-B1H method for which 45 different experimental conditions were tested: five time points and three different IPTG and 3-AT concentrations. We find that GRaMS analysis is robust to the different experimental parameters whereas other analysis methods give widely varying results depending on the conditions of the experiment. Finally, we demonstrate that the CV-B1H assay can be performed in liquid media, which produces recognition models that are similar in quality to sequences recovered from selection on solid media. © 2011 The Author(s).


Gupta A.,Program in Gene Function and Expression | Meng X.,Program in Gene Function and Expression | Meng X.,Sangamo BioSciences | Zhu L.J.,Program in Gene Function and Expression | And 4 more authors.
Nucleic Acids Research | Year: 2011

Zinc finger nucleases (ZFNs) facilitate tailor-made genomic modifications in vivo through the creation of targeted double-stranded breaks. They have been employed to modify the genomes of plants and animals, and cell-based therapies utilizing ZFNs are undergoing clinical trials. However, many ZFNs display dose-dependent toxicity presumably due to the generation of undesired double-stranded breaks at off-target sites. To evaluate the parameters influencing the functional specificity of ZFNs, we compared the in vivo activity of ZFN variants targeting the zebrafish kdrl locus, which display both high on-target activity and dose-dependent toxicity. We evaluated their functional specificity by assessing lesion frequency at 141 potential off-target sites using Illumina sequencing. Only a minority of these off-target sites accumulated lesions, where the thermodynamics of zinc finger-DNA recognition appear to be a defining feature of active sites. Surprisingly, we observed that both the specificity of the incorporated zinc fingers and the choice of the engineered nuclease domain could independently influence the fidelity of these ZFNs. The results of this study have implications for the assessment of likely off-target sites within a genome and point to both zinc finger-dependent and -independent characteristics that can be tailored to create ZFNs with greater precision. © 2010 The Author(s).

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