The research presented here was supported by the Pennsylvania Department of Health, The Ellison Medical, The V and The Emerald Foundations.

1. Expression of recombinant TcTERT
<ol>
<li>Inoculate 6 L of 2YT media (6 x 2 L Baffled Erlenmeyer Flasks containing 1 L of 2YT broth in each) from six fresh plates of transformed Rosetta(DE3)pLysS cells containing the synthetic TcTERT plasmid with a TEV cleavable N-terminal hexahistidine-tag.</li>
<li>Grow cells at 37&deg;C while shaking at 220 rpm to an OD600 of 0.5-0.6.</li>
<li>Induce protein expression by adding 1 mM IPTG.</li>
<li>Reduce temperature to 30&deg;C, slow shaking to 150 rpm, and allow cells to express protein for 4-5 h.</li>
<li>Harvest cells by centrifugation at 4&deg;C and 3500 rpm for 30 min.</li>
<li>Resuspend each 1 L cell pellet in 20 ml of buffer containing 25 mM Tris, pH 7.5, 10% glycerol, 0.5 M KCl, 15 mM Imidazole, 5 mM &beta;-mercaptoethanol, and 0.1 mM phenylmethyl sulphonyl fluoride (PMSF) and benzamidine.</li>
<li>Flash freeze resuspended cell mixture by slowly dripping the cells into a 50 ml plastic conical tube filled with liquid nitrogen so that each drop freezes on impact in the liquid N2. Store frozen cells at -80&deg;C until ready for protein purification.</li>
</ol>
2. Purification of TcTERT
<ol>
<li>Thaw cells at room temperature until a liquid consistency is reached and then move to metal beakers on ice for sonication.</li>
<li>Sonicate cells on ice for 2 min in 30 second intervals with 30 second pauses, at approximately 51 W of power.</li>
<li>Spin down lysed cells at 18,000 rpm for 20 min to separate the soluble and insoluble protein fractions and carefully remove the supernatant for Nickel affinity chromatography.</li>
<li>Equilibrate the Ni-NTA (nickel-nitrilotriacetic acid) column with buffer containing 25 mM Tris, pH 7.5, 10% glycerol, 0.5 M KCl, 15 mM imidazole, 5 mM &#946;-mercaptoethanol, and 0.1 mM PMSF and benzamidine.</li>
<li>Load supernatant over the resin and collect the flow through.</li>
<li>Wash the column with buffer containing 25 mM Tris, pH 7.5, 10% glycerol, 500 mM KCl, 15 mM imidazole, 5 mM &#946;-mercaptoethanol, and 0.1 mM PMSF to remove all residual and non-specifically bound proteins.</li>
<li>Elute TcTERT of the Ni-NTA resin using an imidazole gradient of 15 - 300 mM and collect 3 ml fractions for SDS PAGE analysis.</li>
<li>Use PAGE analysis to determine which fractions contain TcTERT protein and concentrate these fractions in an Amicon 30 kDa (Millipore) filter.</li>
<li>Concentrate TcTERT fractions to a volume less than 10 ml for further purification.</li>
<li>The TcTERT is further purified over the cation-exchange column (HS poros, Applied Biosystems) pre-equilibrated with buffer containing 25 mM Tris, pH 7.5, 10% glycerol, 500 mM KCl, and 1 mM DTT.</li>
<li>Load the protein mix on the HS poros column and wash it to remove all protein and nucleic acid contaminants.</li>
<li>Wash the column with buffer containing 500 mM KCl followed by 700 mM KCl to remove any remaining contaminants.</li>
<li>Elute the TcTERT protein off the HS poros column with 1 M KCl buffer. At this point the protein should be more than 99% pure. </li>
<li>Remove any protein aggregates by passing the protein over a Superdex-200 sizing column (size exclusion chromatography - GE Healthcare), pre-equilibrated with buffer containing 25 mM Tris, pH 7.5, 10% glycerol, 500 mM KCl, and 1 mM TCEP.</li>
</ol>
3. Tribolium castaneum beetle culture
<ol>
<li>The T. castaneum beetles where initially provided as a gift by Dr. S. Brown, Division of Biology, Kansas State University.</li>
<li>Start T. castaneum culture by adding one hundred adult beetles to a container with 28.5 g of flour and 1.5 g of dried baker's yeast.</li>
<li>Store beetle cultures at room temperature and in a dark, humid environment for three to four weeks to allow for reproduction and growth. Place a beaker containing water in the beetle storage area to ensure a humid environment.</li>
<li>Harvest the beetle larvae as needed for RNA isolation. Allow the remaining larvae to mature to adults, keeping them in the same culture.</li>
<li>Every month, transfer the most active beetles (no more than a hundred) into a new flask containing fresh flour and yeast to replenish their food supply.</li>
</ol>
4. T. castaneum Total RNA isolation
<ol>
<li>Harvest 20 beetle larvae from the culture (each culture should contain between 500-1000 larvae) by separating them from adult beetles and the flour/yeast culture. Eliminate flour or yeast particles attached to the beetles before grinding them.</li>
<li>Sterilize the bench area, the mortar, and the pestle (consider treating the gloves as well) used for this procedure with RNaseZap (Ambion, Inc.) to remove all traces of ribonucleases before grinding the beetles.</li>
<li>Grind larvae in liquid N2 into a fine powder using a mortar and pestle and transfer the powder to a 50 ml centrifuge tube while maintaining the liquid N2 to ensure that the powder stays frozen until the extraction buffer is added.</li>
<li>Allow excess liquid N2 to evaporate from the powder. After the liquid N2 has evaporated, homogenize the beetle powder in 200 &mu;l of extraction buffer (per 20 beetle larvae) containing 25 mM Tris-HCl, 5 mM &#946;-mercaptoethanol, 1 mM EGTA, 0.1 mM benzamidine, 200 mM KCl, 10% glycerol, 10 mM imidazole and 20U of RNasin, pH 7.5 and incubate on ice for 30 minutes.</li>
<li>Centrifuge the homogenate at 15,000 rpm for 20 minutes at 4&deg;C, collect the supernatant and flash freeze the sample in liquid N2. If necessary, samples can be stored at -80&deg;C overnight.</li>
<li>Extract the total RNA using the RNeasy Mini Kit (Qiagen).</li>
</ol>
5. In vitro reconstitution of Tribolium castaneum telomerase
<ol>
<li>Mix 20 &mu;g of recombinant His-tagged TcTERT with 50 &mu;l of T. castaneum total RNA in the binding buffer containing 25 mM Tris-HCl, 200 mM KCl, 10% glycerol, 5 mM &#946;-mercaptoethanol, and 10 mM imidazole, pH 7.5.</li>
<li>Incubate the TcTERT &#x2013; total RNA mixture for two hours at 22&deg;C in T. castaneum lysate in the presence of RNasin inhibitor to prevent RNA degradation.</li>
<li>Purify the complex over a Ni-NTA column to remove lysate impurities and excess RNA.</li>
</ol>
6. Telomerase repeat amplification protocol (TRAP) assays
<ol>
<li>Mix 4 &mu;g of Ni-NTA purified T. castaneum telomerase in 50 &mu;l elongation buffer containing 20 mM Tris-HCl (pH - 8.3), 7.5 mM MgCl2, 63 mM KCl, 0.05% Tween 20, 1 mM EGTA, 0.01% BSA, 0.5 mM dNTPs, 1 &mu;M Tcas-TS DNA primer (5'-AAGCCGTCGAGCAGAGTC-3'), and incubate the mixture at 30&deg;C for 60 minutes to allow elongation of the Tcas-TS DNA primer by the reconstituted T. castaneum telomerase.</li>
<li>Extract the elongated single-stranded DNA (ssDNA) by adding an equal volume of phenol chloroform to the reaction mixture. Shake the mixture gently until the emulsion is homogeneous.</li>
<li>Centrifuge the sample at 12,000 rpm for five minutes at 4&deg;C to separate the ssDNA from contaminants.</li>
<li>Transfer the top aqueous layer of the sample to a 1.5 ml eppendorf tube; add a final concentration of 10 mM NaOAc (pH 5.0), 1 mM of MgCl2, and 66.4% EtOH and then allow the sample to precipitate overnight at -20&deg;C.</li>
<li>Spin the sample at 15,000 rpm for thirty minutes at 4&deg;C to pellet the precipitated ssDNA. Decant the solution using a pipettor and then add 200 &mu;l of 70% EtOH to the sample to wash remaining NaOAc and MgCl2 from the sample. Centrifuge the sample for five minutes at the same speed and temperature to re-pellet the sample.</li>
<li>Decant the EtOH solution using a pipettor and remove remaining traces of ethanol by allowing the eppendorf tube to incubate open at room temperature for three minutes. Having ethanol present in the sample will interfere with the PCR amplification step.</li>
<li>Resuspend the ssDNA pellet (containing the telomerase elongated product and the Tcas-TS primer) in 50 &mu;l of PCR reaction buffer containing 10 mM Tris-HCl (pH 8), 50 mM KCl, 2 mM MgCl2, 100 &mu;M dNTPs (dATP, dGTP and dTTP), 10 &mu;M [32P] dCTP, 1 &mu;M Tcas-CX primer (5'-GTGTGACCTGACCTGACC-3') and 1.25 U Taq DNA polymerase.</li>
<li>PCR amplify the telomerase elongation products (29 cycles of 94&deg;C for 30 seconds, 50&deg;C for 30 seconds, and 72&deg;C for 1 minute), so that they are visible on a 12% polyacrylamide gel.</li>
<li>Pre-run the
12% gel with Tris-Borate-EDTA (TBE) running buffer containing 44.5 mM Tris, 44.5 mM Boric Acid, 1 mM EDTA, pH 8 for thirty minutes prior to running the PCR samples. 
After pre-running and loading the gel with the PCR reactions, run the gel at 185 Volts for 1.5-2 hours at 4&deg;Celsius or on ice.</li>
<li>Dry the gel and use the phosphor imaging plate to visualize activity.</li>
</ol>
7. Representative Results:
An example of the purified TcTERT after size exclusion chromatography is shown in Figure 2. The protein concentrated after purification is run on a 12% SDS-polyacrylamide gel and is shown to be more than 99% pure (Figure 2A). The size exclusion (S200) chromatogram is also provided (Figure 2B) to demonstrate the purity and lack of aggregates from the purified protein sample. A yield of 5 mg protein after all purification steps have been completed is usually obtained although the final yield can vary.
A representative TRAP assay for the reconstituted T. castaneum telomerase is shown in Figure 3 as previously reported by Mitchell et al.12. The step-wise ladder of telomerase products can be seen in lane 1, however they are absent in both the RNase treated telomerase and the TERT active site mutant (D251A) samples in lanes 2 and 3 respectively.
<img src="/files/ftp_upload/2799/2799fig1.jpg" alt="Figure 1"/> 
Figure 1. Flow chart of the steps involved in the reconstitution of the recombinant TcTERT. First, the recombinant TcTERT is over-expressed in E. coli. The protein is then purified by nickel, cation exchange, and size exclusion chromatography. The T. castaneum beetles are cultured in house and their larvae are used to isolate the total RNA. The purified TcTERT and the total RNA are then mixed to reconstitute the telomerase RNP complex, and a subsequent TRAP assay is used to confirm the RNP complex is an active telomerase.
<img src="/files/ftp_upload/2799/2799fig2.jpg" alt="Figure 2"/> 
Figure 2. Size exclusion chromatogram and SDS PAGE analysis of TcTERT protein. (A) 12% SDS-polyacrylamide gel of the TcTERT protein after size exclusion chromatography. The TcTERT protein (70 kDa) migrates faster on the SDS PAGE gel because it has a high isoelectric point (pI - 9.8). (B) Size exclusion chromatogram (Superdex S200) of the TcTERT protein.
<img src="/files/ftp_upload/2799/2799fig3.jpg" alt="Figure 3"/> 
Figure 3. TRAP assays of the in vitro reconstituted Tribolium castaneum telomerase adapted from Mitchell et al.12. Lane 1: wild type TcTERT and total RNA isolated from beetle larvae. Lane 2: wild type TcTERT and RNase treated larvae total RNA and cell extracts. Lane 3: active site mutant TcTERT (D251A) and beetle larvae total larvae RNA.

<ol>
	<li>Gillis, A. J., Schuller, A. P., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+Tribolium+castaneum+telomerase+catalytic+subunit+TERT.">Structure of the Tribolium castaneum telomerase catalytic subunit TERT.</a> Nature. 455, 633-637 (2008).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+specific+telomere+terminal+transferase+activity+in+Tetrahymena+extracts.">Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.</a> Cell. 43, 405-413 (1985).</li><li>Harley, C. B., Futcher, A. B., Greider, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres+shorten+during+ageing+of+human+fibroblasts.">Telomeres shorten during ageing of human fibroblasts.</a> Nature. 345, 458-460 (1990).</li><li>Hayflick, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Limited+in+vitro+Lifetime+of+Human+Diploid+Cell+Strains.">The Limited in vitro Lifetime of Human Diploid Cell Strains.</a> Exp Cell Res. 37, 614-636 (1965).</li><li>Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres:+no+end+in+sight.">Telomeres: no end in sight.</a> Cell. 77, 621-623 (1994).</li><li>Kim, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+association+of+human+telomerase+activity+with+immortal+cells+and+cancer.">Specific association of human telomerase activity with immortal cells and cancer.</a> Science. 266, 2011-2015 (1994).</li><li>Bodnar, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extension+of+life-span+by+introduction+of+telomerase+into+normal+human+cells.">Extension of life-span by introduction of telomerase into normal human cells.</a> Science. 279, 349-352 (1998).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+telomere+terminal+transferase+of+Tetrahymena+is+a+ribonucleoprotein+enzyme+with+two+kinds+of+primer+specificity.">The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity.</a> Cell. 51, 887-898 (1987).</li><li>Greider, C. W., Blackburn, E. 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+telomeric+sequence+in+the+RNA+of+Tetrahymena+telomerase+required+for+telomere+repeat+synthesis.">A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis.</a> Nature. 337, 331-337 (1989).</li><li>Jacobs, S. A., Podell, E. R., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+essential+N-terminal+domain+of+telomerase+reverse+transcriptase.">Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase.</a> Nat Struct Mol Biol. 13, 218-225 (2006).</li><li>Rouda, S., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+RNA-binding+domain+of+telomerase:+implications+for+RNA+recognition+and+binding.">Structure of the RNA-binding domain of telomerase: implications for RNA recognition and binding.</a> Structure. 15, 1403-1412 (2007).</li><li>Mitchell, M., Gillis, A., Futahashi, M., Fujiwara, H., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+telomerase+catalytic+subunit+TERT+binding+to+RNA+template+and+telomeric+DNA.">Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA.</a> Nat Struct Mol Biol. 17, 513-518 (2010).</li></ol>

No conflicts of interest declared.

The method presented here allows for the production of large amounts of the catalytic subunit of T. castaneum telomerase TERT in soluble, active form for structural and biochemical studies. The method of TcTERT over-expression is sensitive to subtle changes in temperature or cell density from those stated above and can dramatically affect the levels of protein expression. Specifically, we have found that inducing the cells for protein expression before the optical density reaches 0.5 at 600 nm, or lowe...

<table border="1">
 <tbody>
 <tr>
 <th>Name of Reagent</th>
 <th>Company</th>
 <th>Catalog Number</th>
 </tr>
 <tr>
 <td>Rosetta(DE3)plysS Cells</td>
 <td>Novagen</td>
 <td>70956</td>
 </tr>
 <tr>
 <td>2YT Broth</td>
 <td>Teknova</td>
 <td>Y0215</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Gold Biotechnology</td>
 <td>I2481C</td>
 </tr>
 <tr>
 <td>MISONIX Sonicator 3000</td>
 <td>Qsonica, LLC.</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&Auml;KTA Purifier FPLC</td>
 <td>GE Life Sciences </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ni-NTA Superflow Resin</td>
 <td>Qiagen</td>
 <td>30410</td>
 </tr>
 <tr>
 <td>Amicon Ultra-15 Centrifugal Filter Device</td>
 <td>Millipore</td>
 <td>UFC903008</td>
 </tr>
 <tr>
 <td>POROS 50 HS Strong Cation Exchange Packing</td>
 <td>Applied Biosystems</td>
 <td>1-3359-06</td>
 </tr>
 <tr>
 <td>POROS 50 HQ Strong Cation Exchange Packing</td>
 <td>Applied Biosystems</td>
 <td>1-2559-06</td>
 </tr>
 <tr>
 <td>Superdex 200 10/300 Size-Exclusion Column</td>
 <td>GE Life Sciences</td>
 <td>17-5175-01</td>
 </tr>
 <tr>
 <td>Phenol: Chloroform: Isoamyl 25:24:1 with 10mM Tris, pH 8, 1mM EDTA</td>
 <td>Sigma</td>
 <td>P3803-100mL</td>
 </tr>
 <tr>
 <td>RNaseZap</td>
 <td>Ambion</td>
 <td>AM9780</td>
 </tr>
 <tr>
 <td>Recombinant Rnasin Ribonuclease Inhibitor</td>
 <td>Promega</td>
 <td>N251B</td>
 </tr>
 <tr>
 <td>RNeasy Mini Kit</td>
 <td>Qiagen</td>
 <td>74104</td>
 </tr>
 <tr>
 <td>DNA oligonucleotides</td>
 <td>Integrated DNA Technologies</td>
 <td>&nbsp;</td>
 </tr>
 
 </tbody>
</table>

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.

Watch this Scientific Journal Video about In vitro Reconstitution of the Active T. castaneum Telomerase at JoVE.com

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.

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 ...

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11.6K Aufrufe.  University of Pennsylvania. Die Bemühungen um die katalytische Untereinheit der Telomerase zu isolieren, TERT, in ausreichenden Mengen für strukturelle Studien haben mit begrenztem Erfolg seit mehr als einem Jahrzehnt getroffen. Hier präsentieren wir Methoden zur Isolierung des rekombinanten Tribolium castaneum TERT ( Tc TERT) und die Wiederherstellung der aktiven T. castaneum Telomerase ribonucleoprotein (RNP)-Komplex In-vitro-.

Serie: In vitro Reconstitution of the Active T. castaneum Telomerase

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &amp;#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...