העבודה נתמכה על ידי האוניברסיטה של ורונה בתוך המסגרת של פרויקט משותף 2014 (אפיון משפחת הגן ATL הגפן ועל מעורבותו בהתנגדות Plasmopara viticola).

<ol>
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המחברים אין לחשוף.

בעידן הגנומי, משפחות גנים רבים כבר שאפיינו עמוקות של מספר מיני צמחים. מידע זה הינו preliminary to מחקרים פונקציונלי ומספק מסגרת להמשיך לחקור. את התפקיד של חברים שונות במשפחה. בהקשר זה, יש גם צורך ומאפשר מערכת נומנקלטורה כדי לזהות באופן ייחודי לכל חבר במשפחה, הימנעות יתירות בבלבולים שעלולות להתעו?...

במהלך העשור האחרון, מחקרים רבים בהגפן גנומיקה התבצע. הגפן הוא יבול כלכלית הרלוונטיים המוכרים, אשר הפך מודל למחקר על פרי פיתוח ועל התגובות של צמחים וודי מדגיש ביוטיים, והאביוטיים. בהקשר זה, שחרורו של הגנום cv. PN40024 vinifera גפן 20071 וגירסה מעודכנת שלה 20112 הוביל הצטברות מהירה של "טכנולוגיות" סרגל נתונים וכדי פרץ של מחקרים תפוקה גבוהה. בהתבסס על הנתונים שפורסמו רצף, ניתוח מקיף של משפחה גן מסוים (בדרך כלל מורכב של חלבונים שיתוף מוטיבים שנשמרת, הדמיון המבני ו/או פונקציונלי ויחסים אבולוציונית), ניתן כעת לבצע כדי לחשוף את פונקציות מולקולרית, אבולוציה, וכן ביטוי גנים פרופילים. ניתוחים אלה יכול לתרום להבנה כיצד משפחות גנים לשלוט תהליכים פיזיולוגיים ברמת הגנום כולו.
היבטים רבים של מחזור החיים של צמחים מוסדרים על ידי בתיווך אוביקוויטין השפלה של חלבונים מפתח, אשר דורשים מחזור מכויל כדי להבטיח תהליכים תאיים רגיל. חשוב על מרכיבי התהליך בתיווך אוביקוויטין השפלה הם ligases אוביקוויטין E3, אשר אחראים על גמישות מערכת, בזכות הגיוס של מטרות מוגדרות3. בהתאם לכך, אנזימים אלה מייצגים משפחה גן ענק, עם בסביבות 1,400 E3 ליגאז קידוד הגנים לחזות את הגנום תודרנית לבנה 4, כל ליגאז אוביקוויטין E3 הוא ייצג ubiquitination של חלבונים יעד ספציפיות. למרות החשיבות של המצע ספציפי ubiquitination בתקנה התאית בצמחים, מעט מאוד ידוע על איך מוסדר השביל ubiquitination, חלבונים היעד זוהו רק במקרים מעטים. פיענוח של מנגנונים אלה ירידה לפרטים ורגולציה מסתמך קודם על זיהוי ואפיון של הרכיבים השונים של המערכת, במיוחד את ligases E3. בין ligases אוביקוויטין, תת ATL מאופיין על ידי חברי 91 ב לבנה א הצגת טבעת-H2 האצבע תחום5,6, חלקם תפקיד ההגנה, הורמון תגובות7.
הצעד הראשון מכרעת להגדרת חברי משפחה חדשה ג'ין הוא ההגדרה המדויקת של התכונות משפחה, כגון קונצנזוס במוטיבים, תחומים מרכזיים המאפיינים רצף החלבון. אכן, אמין לאחזור של כל גן בני המשפחה מבוסס על ניתוח הפיצוץ דורש מספר מאפיינים רצף חובה, בתחומים חלבון מסוים, אחראי על חלבון פונקציה/פעילות, הגשה חתימת חלבון. זה ניתן בהנחייתם של אפיון הקודם מאותה משפחה ג'ין מיני צמחים אחרים או מושגת על-ידי ניתוח גנים שונים השייכים putatively מאותה משפחה, מינים שונים כדי לבודד רצפים נפוצות. בני המשפחה ואז ניתן בנפרד בשם בעקבות כללים משותפים מיושב על ידי קונסורציומים בינלאומי עבור זן צמחים נתון. בהגפן, למשל, הליך כזה הוא נתון ההמלצות של ועדת המינוח סופר ענבים ג'ין ביאור (sNCGGa), הקמת הבנייה של עץ פילוגנטי כולל נ' vinifera ו- לבנה א גנים בני המשפחה כדי לאפשר ג'ין ביאור בהתבסס על נוקלאוטיד רצפים8.
כרומוזום לוקליזציה של בני המשפחה וסקר שכפול גנטי לאפשר סימון הנוכחות של הגנום כולו או טנדם גנים המשוכפלת. מידע כזה מופיע שימושי לפענח פונקציות בשם ג'ין, מאז עלול להראות יתירות תפקודית או לחשוף למצבים שונים, כלומר, ללא-functionalization, ניאו-functionalization או תת functionalization9. שני ניאו - ולא משנה - functionalization הם אירועים חשובים יוצר חידוש גנטי, מתן רכיבים סלולר חדשים עבור הצמח ההסתגלות שינוי סביבות10. בפרט, כפילויות של אב קדמון גנים וייצור של גנים חדשים היו תכופים מאוד במהלך האבולוציה של הגנום הגפן, שהוקם גנים שמקורם הפרוקסימלית ואת טנדם כפילויות בהגפן היו בסבירות גבוהה יותר לייצר חדש פונקציות11.
פרופיל transcriptomic זה גורם מפתח נוסף לפענח את ג'ין תפקוד המשפחה. הזמינות של מסדי נתונים הציבור נותן גישה כמות עצומה של נתונים transcriptomic אפשר לנצל ולכן כדי להקצות פונקציות בשם ג'ין משפחתם באמצעות בקנה מידה גדול סיליקו ביטוי ניתוחים. ואכן, הביטוי המיוחד של כמה גנים באיברים צמח מסוים או בתגובה לחצים מסוימים יכול לתת כמה רמזים לגבי התפקידים בשם של החלבונים המתאים בתנאים מוגדרים, ולתת תמיכה היפותזות על האפשר functionalization המשנה של גנים המשוכפלת להגיב על אתגרים שונים. למטרה זו, חשוב לקחת בחשבון מספר נתונים (datasets): אלה יכול להיות זמין כבר גנים ביטוי מטריצות, כגון הרי האטלס הגנום כולו transcriptomic של הגפן איברים ושלבים התפתחותיים12, או יכול להיות בנוי אד הוק על ידי מאחזר datasets transcriptomic עבור המין הצמח המסויים נתון מדגיש מוגדרים. יתר על כן, בגישה פשוטה באמצעות שתי מטריצות, אחד עם נתוני הדמיון pairwise והשני עם המקדמים של pairwise ביטוי משותף ניתן להחיל כדי להעריך את קשרי הגומלין בין תבניות דמיון וביטוי רצף בתוך משפחה ג'ין.
מטרת עבודה זו היא לספק גישה גלובלית, הגדרת מבנה הגן, חלבון שנשמרת מוטיבים, מיקום כרומוזומלית, ג'ין כפילויות ותבניות הביטוי, כמו גם נבואתו של אתרי לוקליזציה, זרחון החלבון, להשיג אפיון ממצה של משפחה גנטי בצמחים. גישה כזו כוללת חל כאן אפיון משפחת ATL E3 ליגאז אוביקוויטין בבית הגפן. על פי תפקידם המתרחב של חברי תת ATL בוויסות תהליכים תאיים מפתח7, עבודה זו יכול גם לסייע הזיהוי של מועמדים ללימודים פונקציונלי, ולפצח בסופו של דבר את המנגנונים המולקולריים המסדירים הסתגלות של היבול חשוב על סביבתו.

<table>
	<tbody>
		<tr>
			<td>Personal computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Local Alignment Search Tool (BLAST)</td>
			<td></td>
			<td></td>
			<td>https://blast.ncbi.nlm.nih.gov/Blast.cgi</td>
		</tr>
		<tr>
			<td>Molecular Evolutionary Genetics Analysis (MEGA)</td>
			<td></td>
			<td></td>
			<td>http://www.megasoftware.net/</td>
		</tr>
		<tr>
			<td>Motif-based sequence analysis tools (MEME)</td>
			<td></td>
			<td></td>
			<td>http://meme-suite.org/</td>
		</tr>
		<tr>
			<td>Geneious</td>
			<td>Biomatters Limited</td>
			<td></td>
			<td>http://www.geneious.com/</td>
		</tr>
		<tr>
			<td>ProtParam Tool</td>
			<td></td>
			<td></td>
			<td>http://web.expasy.org/protparam/</td>
		</tr>
		<tr>
			<td>ngLOC</td>
			<td></td>
			<td></td>
			<td>http://genome.unmc.edu/ngLOC/index.html</td>
		</tr>
		<tr>
			<td>TargetP v1.1 Server</td>
			<td></td>
			<td></td>
			<td>http://www.cbs.dtu.dk/services/TargetP/</td>
		</tr>
		<tr>
			<td>Protein Prowler</td>
			<td></td>
			<td></td>
			<td>http://bioinf.scmb.uq.edu.au:8080/pprowler_webapp_1-2/</td>
		</tr>
		<tr>
			<td>MUsite</td>
			<td></td>
			<td></td>
			<td>http://musite.sourceforge.net/</td>
		</tr>
		<tr>
			<td>Pfam</td>
			<td></td>
			<td></td>
			<td>http://pfam.xfam.org/</td>
		</tr>
		<tr>
			<td>TMHMM Server v. 2.0</td>
			<td></td>
			<td></td>
			<td>http://www.cbs.dtu.dk/services/TMHMM/</td>
		</tr>
		<tr>
			<td>ProtScale</td>
			<td></td>
			<td></td>
			<td>http://web.expasy.org/protscale/</td>
		</tr>
		<tr>
			<td>Grape Genome Database (CRIBI)</td>
			<td></td>
			<td></td>
			<td>http://genomes.cribi.unipd.it/grape/</td>
		</tr>
		<tr>
			<td>PhenoGram</td>
			<td></td>
			<td></td>
			<td>http://visualization.ritchielab.psu.edu/phenograms/plot</td>
		</tr>
		<tr>
			<td>MCScanX</td>
			<td></td>
			<td></td>
			<td>http://chibba.pgml.uga.edu/mcscan2/</td>
		</tr>
		<tr>
			<td>Interactive Tree Of Life (iTOL)</td>
			<td></td>
			<td></td>
			<td>http://itol.embl.de/</td>
		</tr>
		<tr>
			<td>UniProt</td>
			<td></td>
			<td></td>
			<td>http://www.uniprot.org/</td>
		</tr>
		<tr>
			<td>Phylogeny.fr</td>
			<td></td>
			<td></td>
			<td>http://www.phylogeny.fr/index.cgi</td>
		</tr>
		<tr>
			<td>MUSCLE</td>
			<td></td>
			<td></td>
			<td>http://www.ebi.ac.uk/Tools/msa/muscle/</td>
		</tr>
		<tr>
			<td>Gblocks Server</td>
			<td></td>
			<td></td>
			<td>http://molevol.cmima.csic.es/castresana/Gblocks_server.html</td>
		</tr>
		<tr>
			<td>Vitis vinifera cv. Corvina gene expression Atlas datamatrix</td>
			<td></td>
			<td></td>
			<td>https://www.researchgate.net/publication/273383414_54sample_datamatrix_geneIDs_Fasoli2012</td>
		</tr>
		<tr>
			<td>Multi Experiment Viewer (MeV)</td>
			<td></td>
			<td></td>
			<td>http://mev.tm4.org/#/welcome</td>
		</tr>
		<tr>
			<td>Sequence Read Archive (SRA)</td>
			<td></td>
			<td></td>
			<td>https://www.ncbi.nlm.nih.gov/sra</td>
		</tr>
		<tr>
			<td>R</td>
			<td></td>
			<td></td>
			<td>https://www.r-project.org/</td>
		</tr>
		<tr>
			<td>EMBOSS Needle (EMBL-EBI)</td>
			<td></td>
			<td></td>
			<td>http://www.ebi.ac.uk/Tools/psa/emboss_needle/</td>
		</tr>
	</tbody>
</table>

comprehensive workflow for the genome-wide identification and expression meta-analysis of the atl e3 ubiquitin ligase gene family in grapevine

סיווג ובמינוח של הגנים במשפחה יכול באופן משמעותי לתרום את התיאור של המגוון של מקודדים חלבונים וכדי נבואתו של פונקציות משפחתי המבוסס על מספר תכונות, כגון הנוכחות של רצף מוטיבים או במיוחד אתרים עבור שינוי post-translational והפרופיל הביטוי של בני המשפחה בתנאים שונים. עבודה זו מתאר פרוטוקול מפורט עבור ג'ין אפיון משפחה. כאן, ההליך חלה על אפיון משפחת ליגאז אוביקוויטין של E3 תודרנית Tóxicos ב Levadura (ATL) בהגפן. השיטות כוללות זיהוי ברמת הגנום של בני המשפחה, אפיון גנים לוקליזציה, מבנה, שכפול, הניתוח של חלבון שנשמרת מוטיבים, התחזית של לוקליזציה חלבון אתרי זירחון וכן ג'ין ביטוי פרופיל על פני המשפחה נתונים (datasets) שונות. הליך כזה, אשר יכול להרחיב עליו ניתוחים נוספים בהתאם למטרות ניסיוני, יכול לחול על משפחה ג'ין כל מיני צמחים אשר זמינים גנומית נתונים, והוא מספק מידע חשוב לזהות את המועמדים מעניין ללימודים פונקציונלי, נותן תובנות המנגנונים המולקולריים של הצמח ההסתגלות לסביבה שלהם.

הגן VIT_05s0077g01970, זיהתה. הכי דומה לבנה א ATL2 (At3g16720) דרך חיפוש BLASTp, שימש בדיקה סקר בני המשפחה ATL הגנום הגפן (cvנ' vinifera פינו נואר PN40024). הניתוח PSI-הפיצוץ נפגשות לאחר כמה מחזורים חשיפת רשימת הגנים בשם משפחה ג'ין ATL של הגפן (איור 1 א'). הנוכחות של התחום טבעת-H2 הקנו?...

Watch this Scientific Journal Video about Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine at JoVE.com

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

מאמר זה מתאר הליך זיהוי ואפיון של משפחה ג'ין הגפן חלה על משפחתו של תודרנית Tóxicos ב Levadura (ATL) E3 אוביקוויטין ligases.

זרימת עבודה מקיפה עבור הביטוי מטא אנליזה ATL E3 אוביקוויטין ליגאז ג'ין משפחת גפן וזיהוי הגנום כולו

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...

comprehensive-workflow-for-genome-wide-identification-expression-meta

Research

JoVE Journal

Biology

10.4K Views.  Università degli Studi di Verona. מאמר זה מתאר הליך זיהוי ואפיון של משפחה ג'ין הגפן חלה על משפחתו של תודרנית Tóxicos ב Levadura (ATL) E3 אוביקוויטין ligases.

Video: Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

הכנת ראשי בתרביות תאים hematopoietic ממח העצם Murine עבור electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

חוט השדרה Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

שימוש ג&#39;ין Pulser מערכת MXcell electroporation כדי Transfect תאים ראשוניים עם יעילות גבוהה

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

רובוטיקה ניתוח תמונה דינאמית ללימודים של ביטוי גנים ברקמות הצמח

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

שימוש מונה תא אוטומטי כדי לפשט את הביטוי מחקרים ג&#39;ין: siRNA מציאה הביטוי IL 4-Gene תלויים תאים Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

הגנום כולו ניתוח באמצעות שבב זיהוי איזופורם ספציפי מטרות ג&#39;ין

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

ב הכינון במבחנה של ט פעילים castaneum שהטלומרז

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

מחיקות Gene הגנום ב<em&gt; סטרפטוקוקוס sanguinis</em&gt; על ידי תפוקה הגבוהה PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

ניתוח של גלובל RNA סינתזה ברמת התא בודד הבאות היפוקסיה

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

הקרנת siRNA לזיהוי יוביקוויטין ורגולטורים מערכת דמויות יוביקוויטין של מסלולים ביולוגיים בתאי יונקים בתרבית

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...