עבודה זו מומנה על ידי מענקי מחקר NIH K01AR053210, R01AR061484 ו מענק מהקרן הלאומית פסוריאזיס.

<ol>
	<li>He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progranulin+is+a+mediator+of+the+wound+response.">Progranulin is a mediator of the wound response.</a> Nat. Med. 9, 225-229 (2003).</li><li>Kessenbrock, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteinase+3+and+neutrophil+elastase+enhance+inflammation+in+mice+by+inactivating+antiinflammatory+progranulin.">Proteinase 3 and neutrophil elastase enhance inflammation in mice by inactivating antiinflammatory progranulin.</a> J. Clin. Invest. 118, 2438-2447 (2008).</li><li>Zhu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+proepithelin+to+epithelins:+roles+of+SLPI+and+elastase+in+host+defense+and+wound+repair.">Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair.</a> Cell. 111, 867-878 (2002).</li><li>Yin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exaggerated+inflammation,+impaired+host+defense,+and+neuropathology+in+progranulin-deficient+mice.">Exaggerated inflammation, impaired host defense, and neuropathology in progranulin-deficient mice.</a> J. Exp. Med. 207, 117-128 (2010).</li><li>Van Damme, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progranulin+functions+as+a+neurotrophic+factor+to+regulate+neurite+outgrowth+and+enhance+neuronal+survival.">Progranulin functions as a neurotrophic factor to regulate neurite outgrowth and enhance neuronal survival.</a> J. Cell. Biol. 181, 37-41 (2008).</li><li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+progranulin+cause+tau-negative+frontotemporal+dementia+linked+to+chromosome+17.">Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17.</a> Nature. 442, 916-919 (2006).</li><li>Cruts, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Null+mutations+in+progranulin+cause+ubiquitin-positive+frontotemporal+dementia+linked+to+chromosome+17q21.">Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21.</a> Nature. 442, 920-924 (2006).</li><li>Guo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulin-epithelin+precursor+binds+directly+to+ADAMTS-7+and+ADAMTS-12+and+inhibits+their+degradation+of+cartilage+oligomeric+matrix+protein.">Granulin-epithelin precursor binds directly to ADAMTS-7 and ADAMTS-12 and inhibits their degradation of cartilage oligomeric matrix protein.</a> Arthritis Rheum. 62, 2023-2036 (2010).</li><li>Feng, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulin+epithelin+precursor:+a+bone+morphogenic+protein+2-inducible+growth+factor+that+activates+Erk1/2+signaling+and+JunB+transcription+factor+in+chondrogenesis.">Granulin epithelin precursor: a bone morphogenic protein 2-inducible growth factor that activates Erk1/2 signaling and JunB transcription factor in chondrogenesis.</a> FASEB. J. 24, 1879-1892 (2010).</li><li>Xu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+oligomeric+matrix+protein+associates+with+granulin-epithelin+precursor+(GEP)+and+potentiates+GEP-stimulated+chondrocyte+proliferation.">Cartilage oligomeric matrix protein associates with granulin-epithelin precursor (GEP) and potentiates GEP-stimulated chondrocyte proliferation.</a> J. Biol. Chem. 282, 11347-11355 (2007).</li><li>Liu, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ADAMTS-7+and+ADAMTS-12+in+the+pathogenesis+of+arthritis.">The role of ADAMTS-7 and ADAMTS-12 in the pathogenesis of arthritis.</a> Nat. Clin. Pract. Rheumatol. 5, 38-45 (2009).</li><li>Liu, C., Dib-Hajj, S. D., Waxman, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Waxman,+S.G.+Fibroblast+growth+factor+homologous+factor+1B+binds+to+the+C+terminus+of+the+tetrodotoxin-resistant+sodium+channel+rNav1.9a+(NaN).">Waxman, S.G. Fibroblast growth factor homologous factor 1B binds to the C terminus of the tetrodotoxin-resistant sodium channel rNav1.9a (NaN).</a> J. Biol. Chem. 276, 18925-18933 (2001).</li><li>Liu, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ADAMTS-7:+a+metalloproteinase+that+directly+binds+to+and+degrades+cartilage+oligomeric+matrix+protein.">ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein.</a> FASEB. J. 20, 988-990 (2006).</li><li>Tang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Growth+Factor+Progranulin+Binds+to+TNF+Receptors+and+Is+Therapeutic+Against+Inflammatory+Arthritis+in+Mice.">The Growth Factor Progranulin Binds to TNF Receptors and Is Therapeutic Against Inflammatory Arthritis in Mice.</a> Science. 332, 478-484 (2011).</li><li>Wu, H., Siegel, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progranulin+Resolves+Inflammation.">Progranulin Resolves Inflammation.</a> Science. 332, 427-428 (2011).</li><li>Hollenberg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+new+family+of+tissue-specific+basic+helix-loop-helix+proteins+with+a+two-hybrid+system.">Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.</a> Mol. Cell. Biol. 15, 3813-3822 (1995).</li><li>Vojtek, A. B., Hollenberg, S. M., Cooper, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+Ras+interacts+directly+with+the+serine/threonine+kinase+Raf.">Mammalian Ras interacts directly with the serine/threonine kinase Raf.</a> Cell. 74, 205-214 (1993).</li></ol>

אין ניגודי אינטרסים הכריז.

שני היברידית ההקרנה שמרים הוכיחה להיות כלי יעיל בזיהוי אינטראקציה חלבון 16, 17. לעומת גישות אחרות לזיהוי חלבונים מחייב שותפים, כגון שיתוף טיהור חלבון שבבי ביוכימיים, שמרים שני מערכת היברידית היא גישה גנטי רגיש, שניתן להשתמש בהם להקרנה מספרים גבוהים מאוד של קידוד ...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td></tr><tr><td> ProQuest שני היברידית מערכת </td><td> Invitrogen </td><td> 10835 </td></tr><tr><td> YPD בינוני צמיחה </td><td> Clontech </td><td> 630409 </td></tr><tr><td> YPD בינוני אגר </td><td> Clontech </td><td> 630410 </td></tr><tr><td> מאגר מינימלי SD אגר </td><td> Clontech </td><td> 630412 </td></tr><tr><td> Leu-DO מוסף </td><td> Clontech </td><td> 630414 </td></tr><tr><td> -Leu/-Trp DO מוסף </td><td> Clontech </td><td> 630417 </td></tr><tr><td> -His/-Leu/-Trp/-Ura DO מוסף </td><td> Clontech </td><td> 630425 </td></tr><tr><td> 3-אמינו-1-,2,4 triazole (3AT) </td><td> Sigma-Aldrich </td><td> A8056 </td></tr><tr><td> מרק לוריא (LB) </td><td> Sigma-Aldrich </td><td> L3022 </td></tr><tr><td> X-Gal </td><td> Invitrogen </td><td> 15520-034 </td></tr><tr><td> סלמון sonicated זרע DNA </td><td> Stratagene </td><td> 201190 </td></tr><tr><td> אמפיצילין </td><td> AMRESCO </td><td> 0339 </td></tr><tr><td> Kanamycin סולפט </td><td> Invitrogen </td><td> 11815-024 </td></tr><tr><td> יעילות Subcloning ™ DH5α ™ תאים המוסמכת </td><td> Invitrogen </td><td> 18265-017 </td></tr><tr><td> Trizma ® בסיס </td><td> Sigma-Aldrich </td><td> T6066 </td></tr><tr><td> ליתיום אצטט </td><td> Sigma-Aldrich </td><td> L4158 </td></tr><tr><td> Ethylenediaminetetraacetic חומצה </td><td> Sigma-Aldrich </td><td> ED </td></tr><tr><td> Nitrocellulose ממברנה </td><td> Bio-Rad </td><td> 162-0115 </td></tr><tr><td> 10 ס&quot;מ צלחת פטרי </td><td> ITI מדעי </td><td> CT-903 </td></tr><tr><td> אינקובטור (30 ° C) </td><td> ATR (Ecotron) </td><td></td></tr></table>

modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin

Progranulin (PGRN), הידוע גם בשם מבשר epithelin granulin (GEP), הוא גורם-593 חומצות אמיניות צמיחה autocrine. PGRN ידוע לשחק תפקיד קריטי במגוון של תהליכים פיזיולוגיים ומחלות, כולל העובר מוקדם, ריפוי פצעים 1, דלקת 2, 3, ו מארח הגנה 4. PGRN גם פונקציות כגורם neurotrophic 5, מוטציות בגן PGRN שגורם לאובדן חלקי של דמנציה לגרום חלבון PGRN frontotemporal 6, 7. מחקרים אחרונים שלנו הביאו לבידודה של PGRN כמו וסת חשוב של התפתחות סחוס השפלה 8-11. למרות PGRN, גילה כמעט לפני שני עשורים, משחק תפקידים מכריעים מצבים פיזיולוגיים ופתולוגיים מרובים, מאמץ לנצל את הפעולות של PGRN ולהבין את המנגנונים המעורבים כבר הקשו באופן משמעותי על ידי חוסר היכולת שלנו לזהות את הקולטן שלו מחייב (ים). כדי לטפל בבעיה זו, פיתחנו מודיfied שמרים שני היברידית (MY2H) המבוססת על המערכת הנפוצה ביותר GAL4 2-היברידית מבוססת. בהשוואה למסך רגיל שמרים שני היברידית, MY2H דרמטי מקצרת את תהליך המסך מפחית את מספר שיבוטים חיובי כוזב. בנוסף, גישה זו היא לשחזור אמין, ויש לנו בהצלחה המועסקים במערכת זו לבודד את החלבונים המחייב של פיתיונות שונים, כולל יון ערוץ 12, תאי חלבון מטריקס 10, 13, 14 ו - גורם גדילה. במאמר זה, אנו מתארים את הליך הניסוי MY2H בפירוט באמצעות PGRN כדוגמה שהוביל לזיהוי TNFR2 כמו הקולטן first PGRN הקשורים ידוע 14, 15.

Watch this Scientific Journal Video about Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin at JoVE.com

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

אנחנו צריכים לשנות את השמרים קונבנציונאלי שני היברידית ההקרנה, כלי גנטי יעיל בזיהוי אינטראקציה חלבון. שינוי זה ניכר מקצרת את התהליך, מפחית את עומס העבודה, והכי חשוב, מפחיתה את מספר תוצאות חיוביות שגויות. בנוסף, גישה זו היא לשחזור ואמין.

השתנה שמרים-Two-Hybrid System כדי לזהות חלבונים אינטראקציה עם גורם הגדילה Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

modified-yeast-two-hybrid-system-to-identify-proteins-interacting

Research

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Biology

28.6K Views.  NYU Hospital for Joint Diseases. אנחנו צריכים לשנות את השמרים קונבנציונאלי שני היברידית ההקרנה, כלי גנטי יעיל בזיהוי אינטראקציה חלבון. שינוי זה ניכר מקצרת את התהליך, מפחית את עומס העבודה, והכי חשוב, מפחיתה את מספר תוצאות חיוביות שגויות. בנוסף, גישה זו היא לשחזור ואמין.

Video: Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads.

פקטור מצופה צמיחה השמה ביד על Explants forebrain הגבי

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

הצבת פקטור מצופה חרוזים צמיחה על עוברי תרנגולת בשלב מוקדם

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

הכנת פקטור T צמיחת תאים מן Splenocytes עכברוש

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Proteomics כדי לזהות חלבונים אינטראקציה עם ליגנד P2X2-מגודרת ערוצי קטיון

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-יוביקוויטין ממברנה שמרים בהתבסס שני היברידי (מיתוס) מערכת: כלי רב עוצמה עבור זיהוי חלבונים אינטראקציות

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - שיטה לזהות Transcriptome רחב אתרי קישור של חלבונים RNA הכבילה

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi הקרנת לזהות פנוטיפים Postembryonic ב ג elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

הפוך מערכת דו היברידית שמרים לזהות יונקים הגרעיני קולטן שאריות כי אינטראקציה עם ligands ו / או יריבים

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

ספקטרומטריית מתחם מיסת לכידה - כלי רב עוצמה לזיהוי הרומן ג-di-GMP חלבונים מפעיל

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis כמודל לזיהוי תרגום ירידת ערך

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...