This research was supported by a grant to RHG from the National Science Foundation, DBI-0445967.

1. Prepare a list of names for genes that you believe are co-regulated for analysis by SCOPE.
Save the list as a text file or copy it to the clipboard to paste into SCOPE in step 3. The file should contain one gene name per line with no additional information. Alternatively, you can prepare the list as a FASTA file containing the actual sequences to be analyzed.
2. Start your web browser and connect to the URL: <a href="http://genie.dartmouth.edu/SCOPE/">http://genie.dartmouth.edu/SCOPE/</a>
3. Enter the information that SCOPE needs to perform the analysis. 
The initial SCOPE page is shown in Figure 1. Different sections are addressed in this step.
<ol>
	<li>Use the 'Species' popup menu to choose the species you will be examining. It is important to choose the correct species because SCOPE refers to the genome to calculate background frequencies of occurrence for any candidate motif it is examining.</li>
 <li>Use the 'upstream sequence" radio buttons to choose either intergenic or fixed length. Intergenic will analyze all the sequence between of the gene you are looking at and the previous (upstream) gene. This will mean that different upstream lengths will be used for each gene. Choosing fixed length will look at exactly that number of nucleotides upstream from the start of the current gene. In this case, SCOPE will examine the same length of upstream sequence for each gene, even if that extends into the previous gene (or not). Typically, 800 nts is the best length to choose, but this can vary with species.</li>
 <li>Next tell SCOPE what gene set to analyze either by pasting in your gene list into the gene list text box, or by pressing the 'choose file' button to select the file containing the list of genes that you created earlier. You may, alternatively, paste in a FASTA sequence file into the same text box.</li>
 <li>The next section of the page contains a checkbox for 'Examine genome for other genes containing found motif(s)?' This option can add considerable analysis time since SCOPE has to evaluate every other gene in the genome. However, this can be very useful in identifying other genes that are good candidates for being co-regulated with the genes in the starting gene set. Since SCOPE analyses are relatively quick, it is suggested that you leave this off in your initial analysis. It can always be turned on from the results page to rerun the analysis, as explained in the results section.</li>
 <li>The 'Results must include' section can be used to enter a motif that you want SCOPE to include in its analysis. You might want to do this if you are looking for a specific motif.</li>
 <li>The last section on the page can be used to enter your email address and a comment to be saved with the analysis. If this is filled in, SCOPE will send an email with a link back to the web page containing results, and it will also include two attachments. One is a plain text file that has all the analysis results in human readable format. The second attachment contains an XML file that has every result that SCOPE has found in a computer readable format. If you want to do some additional analysis on the results, the XML file is very useful. Both files are "zipped" before being sent with the email.</li>
 <li>For this demo, we will start with the same information. This can be easily achieved by pressing the 'Sample Search' button which will fill in the necessary information. Press this button now. Three genes will be entered for you and appropriate choices made for the other fields. Leave these as they are set. The three genes are involved in telomere maintenance in Saccharomyces cerevisiae. The filled in form is shown in Figure 2. Press the 'Run SCOPE' button at the bottom of the page to start the analysis.</li>
</ol>
4. Representative Results: 
The main results of the analysis are shown in Figure 3. The top of the page contains a table of information about the motifs that were found by SCOPE. The first column contains a list of motifs that were found and small colored squares serve as a legend for the graphical motif map shown below. The display of any given motif may be toggled on or off by clicking in the colored box (or where the colored box would be). This can be very useful to hide the display of highly repeated motifs that might make it difficult to see the less prevalent motif patterns. 
Other columns of data are Count (the number of occurrences of that motif in the entire gene set), Sig value (an indication of the significance of that motif), Coverage (the percentage of the submitted genes that contain at least one instance of that motif), and Algorithm (which of the three component algorithms was used to detect the motif).
Clicking on any of the listed motifs will take the user to a page containing detailed information for that motif. The results details are shown for the cyan motif (atgnnnnttg) in Figure 4. On this page, the motif is represented in three ways: a sequence logo, a position weight matrix, and a list of all motif instances with their positions, strands and genes.
A little further down the page are some additional details about the results of looking for other genes containing this motif. As can be seen, in this case there were 1344 other genes containing the motif, all of which actually improved the Sig value when added to the original gene set. Pressing 'Add checked genes to search' will return to the SCOPE setup page with these genes added to the original gene set and the parameters set as they were previously. In this case, 10 extra genes are added to the original three.
Figure 5 shows the results of the analysis containing the extra genes for this motif. The original three genes are on the bottom of the results (in lower case). Looking at the pattern of motifs in the upstream region of these extra genes clearly shows that they are similar. In fact, many of these genes are involved in telomere maintenance as were the original three genes. Note also that the original motif is now the highest scoring motif in this set.
Another set of SCOPE results is shown in Figure 6. In this case, the set of genes are those that are involved in ribosome biogenesis in Saccharomyces cerevisiae. These genes are not actually part of the ribosome but are responsible for assembling ribosomes and include a number of modification enzymes. What is clear in the figure is that the red and green motifs form a reliable pattern that is likely to be involved in regulation of the genes in this set. We are investigating this pattern of "modules" in more detail and will report on it in a later publication.
<img src="/files/ftp_upload/2703/2703fig1.jpg" alt="Figure 1"/> 
Figure 1. Main SCOPE input page. This page is used to enter the genes to be analyzed and to define the species and the length of upstream region to be examined. Optionally, the user can request the results by email or restrict the search to any specified motif. Video help is also available.
<img src="/files/ftp_upload/2703/2703fig2.jpg" alt="Figure 2"/> 
Figure 2. Main SCOPE input page with values filled in for performing a search. These parameters are the result of pressing the 'Sample Search' button. In this case, the check box to find other genes containing the motifs found by SCOPE is checked. This option takes longer to compute (every gene in the genome has to be examined) but can provide interesting insights.
<img src="/files/ftp_upload/2703/2703fig3.jpg" alt="Figure 3"/> 
Figure 3. Main SCOPE results page. This page summarizes the results of the SCOPE search. A list of all high scoring motifs is provided and a color coded motif map shows the positioning of the identified motifs in the set of genes analyzed. Clicking on a colored box next to a motif will toggle the display of that motif on or off in the motif map. In addition to a significance score (Sig value), the fraction of genes containing the motif (coverage), and the algorithm used to find that motif are also provided.
<img src="/files/ftp_upload/2703/2703fig4_1.jpg" alt="Figure 4 top"/> 
<img src="/files/ftp_upload/2703/2703fig4_2.jpg" alt="Figure 4 bottom"/> Figure 4. This results detail page is brought up when a specific motif is clicked in the main results page. It shows details of the individual motif. The sequence logo, the position weight matrix, and the consensus sequence each represent a different kind of summary of the list of motif instances also on the page. Since 'find extra genes' was checked in the original search setup, there is also information on this page about any other genes in the genome that contain this motif. From this page it is also possible to start another SCOPE run including the extra genes identified on this page.

<img src="/files/ftp_upload/2703/2703fig5.jpg" alt="Figure 5"/> Figure 5. This figure shows the results of looking for extra genes for the motif 'atgnnnnttg' shown in Figure 4. The original three genes are in lower case at the bottom of the motif map. The additional genes are shown in upper case. There is a clear pattern to the motifs in the upstream regions of these genes. Notice also that the specified motif shows an algorithm as 'LOOKUP' because that is how it was identified. It actually matches the 5th motif found by SPACER in this analysis.

<img src="/files/ftp_upload/2703/2703fig6.jpg" alt="Figure 6"/> Figure 6. SCOPE output for genes involved in ribosome biogenesis in Saccharomyces cerevisiae. Note the conserved pattern of modules consisting of the motifs 'aaawtttbh' (red) and 'abctcatcd' (green) separated by about 10-30 nts and present at 100-200 nucleotides upstream of transcription start for the gene.

<ol>
	<li>Chakravarty, A., Carlson, J. M., Khetani, R. S., Gross, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+ensemble+learning+method+for+de+novo+computational+identification+of+DNA+binding+sites.">A novel ensemble learning method for de novo computational identification of DNA binding sites.</a> BMC Bioinformatics. 8, 249-249 (2007).</li><li>Carlson, J. M., Chakravarty, A., DeZiel, C. E., Gross, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCOPE:+a+web+server+for+practical+de+novo+motif+discovery.">SCOPE: a web server for practical de novo motif discovery.</a> Nucleic Acids Res. 35, 259-264 (2007).</li><li>Blom, E. J., Roerdink, J. B., Kuipers, O. P., Hijum, S. A. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MOTIFATOR:+detection+and+characterization+of+regulatory+motifs+using+prokaryote+transcriptome+data.">MOTIFATOR: detection and characterization of regulatory motifs using prokaryote transcriptome data.</a> Bioinformatics. 25, 550-551 (2009).</li><li>Blom, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DISCLOSE+:+DISsection+of+CLusters+Obtained+by+SEries+of+transcriptome+data+using+functional+annotations+and+putative+transcription+factor+binding+sites.">DISCLOSE : DISsection of CLusters Obtained by SEries of transcriptome data using functional annotations and putative transcription factor binding sites.</a> BMC Bioinformatics. 9, 535-535 (2008).</li><li>Bushey, A. M., Ramos, E., Corces, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+subclasses+of+a+Drosophila+insulator+show+distinct+and+cell+type-specific+genomic+distributions.">Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions.</a> Genes Dev. 23, 1338-1350 (2009).</li><li>Znaidi, S. <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+the+Candida+albicans+Cap1p+regulon.">Identification of the Candida albicans Cap1p regulon.</a> Eukaryot Cell. 8, 806-820 (2009).</li><li>Sharma, D., Mohanty, D., Surolia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RegAnalyst:+a+web+interface+for+the+analysis+of+regulatory+motifs,+networks+and+pathways.">RegAnalyst: a web interface for the analysis of regulatory motifs, networks and pathways.</a> Nucleic Acids Res. 37, W193-W201 (2009).</li><li>Znaidi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomewide+location+analysis+of+Candida+albicans+Upc2p,+a+regulator+of+sterol+metabolism+and+azole+drug+resistance.">Genomewide location analysis of Candida albicans Upc2p, a regulator of sterol metabolism and azole drug resistance.</a> Eukaryot Cell. 7, 836-847 (2008).</li><li>Carlson, J., Chakravarty, A., Gross, R. B. E. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+beam+search+algorithm+for+the+identification+of+cis-regulatory+elements+in+groups+of+genes.">A beam search algorithm for the identification of cis-regulatory elements in groups of genes.</a> J Comput Biol. 13, 686-701 (2006).</li><li>Carlson, J., Chakravarty, A., Khetani, R., Gross, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bounded+search+for+de+novo+identification+of+degenerate+cis-regulatory+elements.">Bounded search for de novo identification of degenerate cis-regulatory elements.</a> BMC Bioinformatics. 7, 254-254 (2006).</li><li>Chakravarty, A., Carlson, J. M., Khetani, R. S., DeZiel, C. E., Gross, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPACER:+identification+of+cis-regulatory+elements+with+non-contiguous+critical+residues.">SPACER: identification of cis-regulatory elements with non-contiguous critical residues.</a> Bioinformatics. 23, 1029-1031 (2007).</li></ol>

No conflicts of interest declared.

SCOPE provides the researcher with a powerful tool to use for the identification of potential regulatory motifs in sets of coordinately regulated genes. The user is not required to guess at the size of the motif or the number of occurrences of the motif as many other motif finding sites require. These parameters are basically unknowable until the motif is identified. The interface is very simple both for entering sequences or gene names and for viewing the output.
SCOPE output provides detailed information about all of the motifs that are identified, using three different ways of motif representation. Each instance of the motif in all of the genes is listed with position and "strand" information. Graphical results in the form of motif maps provide a visual display that is easy to understand and provides an intuitive way to see patterns in the motifs that are present.

SCOPE is very robust to the presence of noise in the data. Typically, this takes the form of extra genes being present in the starting set that might not actually be co-regulated with the rest of the genes. This often happens when starting with genes that are co-expressed in microarray experiments. Sometimes the experiment is noisy, or there may be several transcription factors activated in the experimental conditions used for the microarray experiment. These different transcription factors will likely have different target sites on the DNA. Even in the presence of 4-fold extraneous genes (noise:signal ratio is 4:1), SCOPE is still maintains 50% of its accuracy in predicting sites1.

Although SCOPE contains over 2 million synonyms for gene names, it sometimes fails to identify some genes names. We are constantly updating our synonym lists, but sometimes find that different synonyms refer to the same gene. In those cases, we do not include the synonyms because of the ambiguity. if you have a gene name that is not found by SCOPE, it is recommended that you refer to the genome specific site to find an alternative gene name to use in SCOPE. Examples of appropriate gene names for each species are provided by SCOPE.
SCOPE currently contains 72 species with new species being added all the time. The web site contains video help as well as FAQs. Source code is freely available to academic users by writing to RHG.

using scope to identify potential regulatory motifs in coregulated genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

Watch this Scientific Journal Video about Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes at JoVE.com

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

تستخدم لتحديد نطاق الدوافع المحتملة في تنظيم الجينات Coregulated

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

using-scope-to-identify-potential-regulatory-motifs-coregulated

Research

JoVE Journal

Biology

10.3K Views.  Dartmouth College. A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

Video: Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

Regulatory T cells: Therapeutic Potential for Treating Transplant Rejection and Type I Diabetes

التنظيمية خلايا T : الإمكانات العلاجية لعلاج رفض الأعضاء والنوع الأول من مرض السكري

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

تطبيق الفحص جيم ايليجانس الدوبامين العصبية للتحلل من التحقق من صحة جينات مرض باركنسون المحتملة

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

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.

الجينوم على نطاق التحليل باستخدام رقاقة لتحديد أهداف محددة الجينات شكل الإسوي

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

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

مناعي لونين الفحص الجينات الأنسجة الخاصة باستخدام أجنة فئران في مرحلة مبكرة

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

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.

[رني الفرز لتحديد الظواهر تال للمرحلة الجنينية في C. ايليجانس</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 ...

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics: الكشف عن التغيرات العددية والهيكلية في جميع الكروموسومات 24 الإنسان وقت واحد باستخدام رواية الفحص FISH OctoChrome

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

عزلة البروتينية ريبوسوم الوليدة منضم<em&gt; في المختبر</em&gt; لتحديد المواقع وقفة بالحركة على طول مرنا

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

باستخدام الحمض النووي الريبي التي تتوسط استراتيجية التغذية التدخل للكشف عن الجينات المسؤولة عن تنظيم حجم الجسم في نيماتودا<em&gt; C. ايليجانس</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

A الكمي الفحص لدراسة البروتين: تفاعلات الحمض النووي، اكتشاف المنظمين الترانسكربتي التعبير الجيني، وتحديد رواية وكلاء المضادة للورم

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus&#160;). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3&#39;-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

A whole-mount immunohistochemical approach, to visualize neurofilament protein expression in the extrahepatic biliary tract in Suncus murinus. is presented here. This protocol can be used to analyze the innervation of all visceral organs in S. murinus or other species.

باستخدام كامل-- جبل المناعية طريقة لدراسة التعصيب من القناة الصفراوية في<em&gt; سونكوس مورينوس</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...