Name: in vitro transcription assays and their application in drug discovery
Uploaded: 2016-09-20T08:00:00
Description: Watch this Scientific Journal Video about In Vitro Transcription Assays and Their Application in Drug Discovery at JoVE.com

This work acknowledges a Faculty Early Career Grant from the University of Newcastle (CM).

Caution: Experiments involve the use of the radioactive isotope 32P and no work should be undertaken until all appropriate safety conditions have been met. Generally personnel are required to attend a safety course and undergo supervised practice prior to experiments using radioactive reagents. Please wear personal protection equipment (thermoluminescent dosimeter, safety glasses, gloves, radiation room lab coats, full length pants, closed-toe shoes) when performing the reaction.
1. Preparation of Assay M...

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Microbiol. 4 (2), 126-131 (2001).</li><li>Gusarov, I., Nudler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+intrinsic+transcription+termination+by+N+and+NusA:+the+basic+mechanisms.">Control of intrinsic transcription termination by N and NusA: the basic mechanisms.</a> Cell. 107 (4), 437-449 (2001).</li><li>Ha, K. S., Toulokhonov, I., Vassylyev, D. G., Landick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NusA+N-terminal+domain+is+necessary+and+sufficient+for+enhancement+of+transcriptional+pausing+via+interaction+with+the+RNA+exit+channel+of+RNA+polymerase.">The NusA N-terminal domain is necessary and sufficient for enhancement of transcriptional pausing via interaction with the RNA exit channel of RNA polymerase.</a> J. Mol. 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S., Landick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-overexpression+of+Escherichia+coli+RNA+polymerase+subunits+allows+isolation+and+analysis+of+mutant+enzymes+lacking+lineage-specific+sequence+insertions.">Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions.</a> J. Biol. Chem. 278 (14), 12344-12355 (2003).</li><li>Ma, C., Yang, X., Lewis, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+transcription+inhibitor+of+RNA+polymerase+holoenzyme+formation+by+structure-based+drug+design:+From+in+silico+screening+to+validation.">Bacterial transcription inhibitor of RNA polymerase holoenzyme formation by structure-based drug design: From in silico screening to validation.</a> ACS Infect. Dis. 2, 39-46 (2016).</li><li>Ma, C., Mobli, M., Yang, X., Keller, A. N., King, G. F., Lewis, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase-induced+remodelling+of+NusA+produces+a+pause+enhancement+complex.">RNA polymerase-induced remodelling of NusA produces a pause enhancement complex.</a> Nucleic Acids Res. 43 (5), 2829-2840 (2015).</li><li>Lewis, P. J., Marston, A. 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In all organisms, transcription is a tightly regulated process. In vitro transcription assays have been developed to provide a platform for testing the effects of transcription factors, small molecules and transcription inhibitors. In this method paper, an assay for general bacterial transcription was described. Transcription assays combined with sequencing gel electrophoresis of transcripts are very important for mechanistic studies as they allow visualization of all the transcription products along a timeline....

Transcription is the process in which RNA is synthesized from a specific DNA template. In eukaryotic cells there are three distinct RNAPs: RNAP I transcribes rRNA precursors, RNAP II is responsible for the synthesis of mRNA and certain small nuclear RNAs, and the synthesis of 5S rRNA and tRNA is performed by RNAP III. In bacteria, there is only one RNAP responsible for the transcription of all classes of RNA. There are three stages of transcription: initiation, elongation and termination. Transcription is one of the most highly regulated processes in the cell. Each stage in the transcription cycle represents a checkpoint for the regulation of gene expression1. For initiation, RNAP has to associate with a sigma factor to form holoenzyme, which is required to direct the enzyme to specific sites called promoters2 to form an open promoter complex. Subsequently, a large suite of transcription factors are responsible for the regulation of RNAP activities during the elongation and termination phases. The transcription factor examined here is the highly conserved and essential protein, NusA. It is involved in regulating transcription pausing and termination, as well as anti-termination during rRNA synthesis3-5.
In vitro transcription assays have been developed as powerful tools to study the complex regulatory steps during transcription6. In general, a linear fragment of DNA including a promoter region is required as the template for transcription. The DNA template is usually generated by PCR or by linearizing a plasmid. Purified proteins and NTPs (including one radiolabeled NTP for detection purposes) are then added and product analyzed following the required period of incubation. Using appropriate templates and reaction conditions, all stages of transcription have been examined using this approach which has enabled detailed molecular characterization of transcription over the last half century7. In combination with information on the 3-dimensional structure of RNAP it has also been possible to probe the molecular mechanism of transcription inhibition by antibiotics and antibiotic leads, and use this information in the development of new, improved drugs8-10.
In this work we provide examples of how transcription assays can be used to determine the mechanism of regulation by transcription elongation/termination factor NusA, and how the mechanism of action of a novel transcription initiation inhibitor lead can be determined.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Obtain the proteins required for transcription assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli RNAP</td>
			<td>Epicentre</td>
			<td>S90250</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparation of DEPC-treated water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>diethyl pyrocarbonate (DEPC)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D5758</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free water&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ambion Nuclease-Free Water</td>
			<td>ThermoFisher</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA template preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard&#160;Plus&#160;SV Minipreps DNA Purification System</td>
			<td>Promega</td>
			<td>A1330</td>
			<td></td>
		</tr>
		<tr>
			<td>ACCUZYME Mix</td>
			<td>Bioline</td>
			<td>BIO-25028</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard SV Gel and PCR Clean-Up System&#160;</td>
			<td>Promega</td>
			<td>A9281</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 3300 fluorospectrometer</td>
			<td>Thermo Scientific</td>
			<td>ND-3300</td>
			<td></td>
		</tr>
		<tr>
			<td>NTP Preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A6559</td>
			<td></td>
		</tr>
		<tr>
			<td>UTP</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>U1006</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G3776</td>
			<td></td>
		</tr>
		<tr>
			<td>CTP</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C9274</td>
			<td></td>
		</tr>
		<tr>
			<td>High Purity rNTPs</td>
			<td>GE Healthcare</td>
			<td>27-2025-01</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-32P UTP&#160;</td>
			<td>PerkinElmer&#160;&#160;</td>
			<td>BLU007C001MC</td>
			<td>Radioactive compound</td>
		</tr>
		<tr>
			<td>RNA ladder preparation&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Novagen Perfect RNA Marker Template Mix 0.1&#8211;1 kb</td>
			<td>Millipore</td>
			<td>69003</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H7006</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>DTT-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 RNAP</td>
			<td>Promega</td>
			<td>P2075</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sequi-Gen GT nucleic acid sequencing cell&#160;</td>
			<td>Bio-Rad&#160;&#160;</td>
			<td>165-3804</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmacote&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>SL2</td>
			<td></td>
		</tr>
		<tr>
			<td>urea&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>U6504</td>
			<td></td>
		</tr>
		<tr>
			<td>tris(hydroxymethyl)aminomethane&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>154563</td>
			<td></td>
		</tr>
		<tr>
			<td>boric acid&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>B7901</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>ED</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide/bis-acrylamide&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A9926</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium persulfate&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#697;&#8242;,N&#697;&#8242;-Tetramethylethylenediamine (TEMED)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N",N"-Tetramethylethylenediamine (TEMED) </td>
			<td>Sigma-Aldrich </td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Transcription Assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>rifampicin&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr>
			<td>formamide&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F9037</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene cyanol&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>X4126</td>
			<td></td>
		</tr>
		<tr>
			<td>heparin&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>84020</td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin Ribonuclease Inhibitor</td>
			<td>Promega</td>
			<td>N2511</td>
			<td></td>
		</tr>
		<tr>
			<td>Transcription buffer&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>DTT-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman&#160;3MM Chr Chromatography Paper</td>
			<td>Fisher Scientific</td>
			<td>05-714-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Radioactive decontaminant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Decon 90</td>
			<td>decon</td>
			<td>decon90</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Treatment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon Trio+ imager</td>
			<td>GE Healthcare Life Sciences</td>
			<td>63-0055-89</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro transcription assays and their application in drug discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

Transcription efficiency can be determined by measuring the level of radiation in the bands at different time points. The pausing assay to test the function of NusA factor enabled visualization of the pause, termination, and run-off products (Figure 1A). In the presence of the N-terminal domain of NusA (NusA NTD; amino acid residues 1-137), appearance of the RNA products was significantly delayed compared to the control experiment lacking NusA. Deletion of amino acid resi...

Watch this Scientific Journal Video about In Vitro Transcription Assays and Their Application in Drug Discovery at JoVE.com

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

The overall goal of this in vitro transcription assay is to study the complex regulatory steps during transcription by providing a platform for testing the effects of transcription factors, small molecules, and transcription inhibitors. This method can help answer key questions in molecular biology, such as the precise mechanisms associated with transcription regulation, the function of transcription factors with unknown mechanisms, and the effects of normal transcription inhibitors. The main advantage of this technique is that it allows direct visualization of all the transcription products along a timeline from a single RNA sequencing gel.Begin this procedure by cleaning the plates from a sequencing gel apparatus twice with 70%ethanol and purified water. Assemble the cell with a comb and two spacers. Dissolve 33.6 grams of urea in 22 milliliters of water and 16.4 milliliters of five times TBE buffer using a magnetic stirrer.Add 16 milliliters of a 40%acrylamide, this acrylamide solution to the urea solution, and filter through a 0.45 micrometer filter. Add to the filtered solutoin 514 microliters of a freshly prepared 10%ammonium persulfate solution. Followed by 51.4 microliters of TEMED.Gently invert the gel solution to avoid excessive aeration and inject immediately and smoothly from the bottom injection hole into the horizontally positioned pre-packed sequencing cell. Take care to avoid making bubbles during the injection. Allow the gel to completely set for one to 1.5 hours before use.To start the procedure for the formation of a transcription elongation complex, add one microliter of RNA polymerase core enzyme to a 1.5 microliter tube. Add one microliter of purified sigma 70 factor to the RNA polymerase and incubate on ice for 10 minutes to form the RNA polymerase holoenzyme. Add to the RNA polymerase holoenzyme 3 microliters of a 500 nano molar purified template DNA in transcription buffer and incubate at 37 degrees Celsius for five minutes to form the open complex.Next, prepare a mixture of reaction components by adding the following reagents to a 1.5 milliliter tube. Five microliters of 10 times transcription buffer, two microliters of 250 micromolar GTP, two microliters of 250 micromolar UTP, one microliter of one millimolar ATP, and one microliter of alpha P 32 labeled UTP. Add DEPC treated water to the mixture to 41 microliters.Transfer the mixture to the tube containing the open complex and incubate at 37 degrees Celsius for fifteen minutes to form the transcription elongation complex stopped at plus 26 nucleotide. After 15 minutes, move the reaction to room temperature and add one microliter of 2.4 milligrams per milliliter rifampicin to a final concentration of 50 micrograms per milliliter. Add one microliter of 0.1 millimolar purified NusA transcription factor to the reaction mixture and incubate for five minutes at room temperature.Add two microliters of 250 micromolars CTP to the reaction mixture to make a final volume of 50 microliters and measure the reaction time using a timer. At each time point, transfer five microliters of the reaction solution into 2 microliters of RNA Gel-loading Buffer to quench the reaction. Prior to electrophoresis of the samples, pre-run the gel for approximately 1.5 hours at 50 degrees Celsius and 50 watts in one time TBE buffer until the temperature of the gel and the buffer is stable at 50 degrees Celsius.When the pre-run is complete, heat the samples at 90 degrees Celsius for 30 seconds and move them onto ice. Load the first well on the top of the gel with two microliters of RNA ladder and then, load the samples into the wells along side the ladder. Run the gel at 50 watts and 50 degrees Celsius in one time TBE buffer for about 1.5 hours, until the bromophenol blue dye reaches about 2/3 down the gel.When the run is complete, slowly open the two gel plates and carefully separate the upper plate from the sequencing cell, taking great care to avoid breaking the gel. Cut chromatography paper to match the gel size from the top to the bromophenol blue dye. Use a Geiger counter to confirm the approximate position of radioactively labeled RNA transcripts.Carefully cover the gel with the filter paper and press firmly until the gel adheres to the filter paper. Cut off and discard the bottom of the gel as radioactive waste, since the unincorporated NTPs run to the bottom of the gel, which can be highly radioactive. Place a similar sized piece of filter paper on the drying bed of a vacuum drying machine and carefully lay the gel-coated paper onto it with the gel side up.Cover the gel with plastic wrap and dry at 60 degrees Celsius for 1.5 hours. After the gel is dry, wrap the other side of the gel-coated filter paper with plastic wrap and place a photostimulable phosphor plate in the imaging case for the appropriate exposure time. Subsequently, image the phosphor plate using an imager scanner according to the manufacturer's instructions.To test the effects of mutant NusA end terminal domain, or NTD in transcription, transcription elongation pausing assays were performed without NusA or with NusA-NTD, NusA-NTD with Helix 3 deleted, and NusA-NTD with alanine substitutions at eight residues. The pause, termination, and run-off products were successfully visualized. In the presence of NusA-NTD, appearance of the RNA products was significantly delayed compared to the control experiment lacking NusA.The pause activities of half-lives of mutant NusA-NTD relative to the wild type protein is shown. Deletion of helix 3, or alternation to alanine of a series of amino acids, resulted in the complete loss of NusA pause activity. Helix 3 residues, arginine 104 and lysine 111, were shown to be essential for NusA-NTD pause activity.In vitro transcription assays can also be used to examine the effects of newly identified bacterial transcription inhibitors, such as compound C5.Percent inhibition of transcription was determined by the RNA product relative to a control against the concentration of C5 used in the reaction. Mechanistically, addition of C5 to RNA polymerase followed by sigma 70 factor to the reaction mixture was significantly more efficient for transcription inhibition than using C5 after the formation of the RNA polymerase holoenzyme. Following this procedure, other methods like ELISA and isothermal titration calorimetry, can be preformed in order to answer additional questions, such as, how a inhibitor affects transcription.Once developed, this technique pave the way for researchers in the field of molecular or chemical biology. To explore the mechanism and related inhibitors in bacterial transcription. Don't forget that working with radioactive reagents can be extremely hazardous and precautions such as full PPE should always be used while preforming this procedure.

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Research

JoVE Journal

Genetics

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Novel Sequence Discovery by Subtractive Genomics

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a larger genomic context. Subtractive genomics enables a researcher to isolate a target sequence of interest (T) by comprehensive sequencing and subtracting out known genetic elements (reference, R). The method can be used to identify novel sequences such as mitochondria, chloroplasts, viruses, or germline restricted chromosomes, and is particularly useful when T cannot be easily isolated from R. Beginning with the comprehensive genomic data (R + T), the method uses Basic Local Alignment Search Tool (BLAST) against a reference sequence, or sequences, to remove the matching known sequences (R), leaving behind the target (T). For subtraction to work best, R should be a relatively complete draft that is missing T. Since sequences remaining after subtraction are tested through quantitative Polymerase Chain Reaction (qPCR), R does not need to be complete for the method to work. Here we link computational steps with experimental steps into a cycle that can be iterated as needed, sequentially removing multiple reference sequences and refining the search for T. The advantage of subtractive genomics is that a completely novel target sequence can be identified even in cases in which physical purification is difficult, impossible, or expensive. A drawback of the method is finding a suitable reference for subtraction and obtaining T-positive and negative samples for qPCR testing. We describe our implementation of the method in the identification of the first gene from the germline-restricted chromosome of zebra finch. In that case computational filtering involved three references (R), sequentially removed over three cycles: an incomplete genomic assembly, raw genomic data, and transcriptomic data.

The purpose of this protocol is to use a combination of computational and bench research to find novel sequences that cannot be easily separated from a co-purifying sequence, which may be only partially known.

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...