Development and validation of a T7 based linear amplification for genomic DNA

BACKGROUND: Genomic maps of transcription factor binding sites and histone modification patterns provide unique insight into the nature of gene regulatory networks and chromatin structure. These systematic studies use microarrays to analyze the composition of DNA isolated by chromatin immunoprecipitation. To obtain quantities sufficient for microarray analysis, the isolated DNA must be amplified. Current protocols use PCR-based approaches to amplify in exponential fashion. However, exponential amplification protocols are highly susceptible to bias. Linear amplification strategies minimize amplification bias and have had a profound impact on mRNA expression analysis. These protocols have yet to be applied to the analysis of genomic DNA due to the lack of a suitable tag such as the polyA tail. RESULTS: We have developed a novel linear amplification protocol for genomic DNA. Terminal transferase is used to add polyT tails to the ends of DNA fragments. Tail length uniformity is ensured by including a limiting concentration of the terminating nucleotide ddCTP. Second strand synthesis using a T7-polyA primer adapter yields double stranded templates suitable for in vitro transcription (IVT). Using this approach, we are able to amplify as little as 2.5 ng of genomic DNA, while retaining the size distribution of the starting material. In contrast, we find that PCR amplification is biased towards species of greater size. Furthermore, extensive microarray-based analyses reveal that our linear amplification protocol preserves dynamic range and species representation more effectively than a commonly used PCR-based approach. CONCLUSION: We present a T7-based linear amplification protocol for genomic DNA. Validation studies and comparisons with existing methods suggest that incorporation of this protocol will reduce amplification bias in genome mapping experiments

Global nucleosome occupancy in yeast

BACKGROUND: Although eukaryotic genomes are generally thought to be entirely chromatin-associated, the activated PHO5 promoter in yeast is largely devoid of nucleosomes. We systematically evaluated nucleosome occupancy in yeast promoters by immunoprecipitating nucleosomal DNA and quantifying enrichment by microarrays. RESULTS: Nucleosome depletion is observed in promoters that regulate active genes and/or contain multiple evolutionarily conserved motifs that recruit transcription factors. The Rap1 consensus was the only binding motif identified in a completely unbiased search of nucleosome-depleted promoters. Nucleosome depletion in the vicinity of Rap1 consensus sites in ribosomal protein gene promoters was also observed by real-time PCR and micrococcal nuclease digestion. Nucleosome occupancy in these regions was increased by the small molecule rapamycin or, in the case of the RPS11B promoter, by removing the Rap1 consensus sites. CONCLUSIONS: The presence of transcription factor-binding motifs is an important determinant of nucleosome depletion. Most motifs are associated with marked depletion only when they appear in combination, consistent with a model in which transcription factors act collaboratively to exclude nucleosomes and gain access to target sites in the DNA. In contrast, Rap1-binding sites cause marked depletion under steady-state conditions. We speculate that nucleosome depletion enables Rap1 to define chromatin domains and alter them in response to environmental cues

Cyclooxygenase-2 enhances α2β1 integrin expression and cell migration via EP1 dependent signaling pathway in human chondrosarcoma cells

<p>Abstract</p> <p>Background</p> <p>Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin (PG) synthase, has been implicated in tumor metastasis. Interaction of COX-2 with its specific EP receptors on the surface of cancer cells has been reported to induce cancer invasion. However, the effects of COX-2 on migration activity in human chondrosarcoma cells are mostly unknown. In this study, we examined whether COX-2 and EP interaction are involved in metastasis of human chondrosarcoma.</p> <p>Results</p> <p>We found that over-expression of COX-2 or exogenous PGE₂increased the migration of human chondrosarcoma cells. We also found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of the COX-2 which was higher than that in normal cartilage. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptors, we discovered that the EP1 receptor but not other PGE receptors is involved in PGE₂-mediated cell migration and α2β1 integrin expression. Furthermore, we found that human chondrosarcoma tissues expressed a higher level of EP1 receptor than normal cartilage. PGE₂-mediated migration and integrin up-regulation were attenuated by phospholipase C (PLC), protein kinase C (PKC) and c-Src inhibitor. Activation of the PLCβ, PKCα, c-Src and NF-κB signaling pathway after PGE₂treatment was demonstrated, and PGE₂-induced expression of integrin and migration activity were inhibited by the specific inhibitor, siRNA and mutants of PLC, PKC, c-Src and NF-κB cascades.</p> <p>Conclusions</p> <p>Our results indicated that PGE₂enhances the migration of chondrosarcoma cells by increasing α2β1 integrin expression through the EP1/PLC/PKCα/c-Src/NF-κB signal transduction pathway.</p

RNA polymerase mapping during stress responses reveals widespread nonproductive transcription in yeast

BACKGROUND: The use of genome-wide RNA abundance profiling by microarrays and deep sequencing has spurred a revolution in our understanding of transcriptional control. However, changes in mRNA abundance reflect the combined effect of changes in RNA production, processing, and degradation, and thus, mRNA levels provide an occluded view of transcriptional regulation. RESULTS: To partially disentangle these issues, we carry out genome-wide RNA polymerase II (PolII) localization profiling in budding yeast in two different stress response time courses. While mRNA changes largely reflect changes in transcription, there remains a great deal of variation in mRNA levels that is not accounted for by changes in PolII abundance. We find that genes exhibiting \u27excess\u27 mRNA produced per PolII are enriched for those with overlapping cryptic transcripts, indicating a pervasive role for nonproductive or regulatory transcription in control of gene expression. Finally, we characterize changes in PolII localization when PolII is genetically inactivated using the rpb1-1 temperature-sensitive mutation. We find that PolII is lost from chromatin after roughly an hour at the restrictive temperature, and that there is a great deal of variability in the rate of PolII loss at different loci. CONCLUSIONS: Together, these results provide a global perspective on the relationship between PolII and mRNA production in budding yeast

Characteristics of virtual unipolar electrograms for detecting isthmus block during radiofrequency ablation of typical atrial flutter

AbstractObjectivesThe purpose of this study was to investigate the characteristics of the second component of local virtual unipolar electrograms recorded at the ablation line during coronary sinus (CS) pacing after radiofrequency ablation (RFA) of the cavotricuspid isthmus (CTI) for typical atrial flutter (AFL).BackgroundRadiofrequency ablation of the CTI can produce local double potentials at the ablation line. The second component of unipolar electrograms represents the approaching wavefront in the right atrium opposite the pacing site. We hypothesized that the morphologic characteristics of the second component of double potentials would be useful in detecting complete CTI block.MethodsRadiofrequency ablation of the CTI was performed in 52 patients (males = 37, females = 15, 62 ± 12 years) with typical AFL. The noncontact mapping system (Ensite 3000, Endocardial Solutions, St. Paul, Minnesota) was used to guide RFA. Virtual unipolar electrograms along the ablation line during CS pacing after RFA were analyzed. Complete or incomplete CTI block was confirmed by the activation sequence on the halo catheter and noncontact mapping.ResultsThree groups were classified after ablation. Group I (n = 37) had complete bidirectional CTI block. During CS pacing, the second component of unipolar electrograms showed an R or Rs pattern. Group II (n = 12) had incomplete CTI block. The second component of unipolar electrograms showed an rS pattern. Group III (n = 3) had complete CTI block with transcristal conduction. The second component of unipolar electrograms showed an rSR pattern.ConclusionsA predominant R-wave pattern in the second component of unipolar double potentials at the ablation line indicates complete CTI block, even in the presence of transcristal conduction

Design, synthesis, cytotoxic activity and molecular docking studies of new 20(S)-sulfonylamidine camptothecin derivatives

20(S)-Sulfonylamidine CPT-derivatives were prepared and tested for cytotoxicity.Several analogs showed superior cytotoxic activity compared to irinotecan.Key structural features related to cytotoxicity were identified by SAR analysis.Compounds 9 and 15c interacted with Topo I-DNA by a different binding mode from CPT.These compounds are new generation CPT-derived antitumor agents.In an ongoing investigation of 20-sulfonylamidine derivatives (9, YQL-9a) of camptothecin (1) as potential anticancer agents directly and selectively inhibiting topoisomerase (Topo) I, the sulfonylamidine pharmacophore was held constant, and a camptothecin derivatives with various substitution patterns were synthesized. The new compounds were evaluated for antiproliferative activity against three human tumor cell lines, A-549, KB, and multidrug resistant (MDR) KB subline (KBvin). Several analogs showed comparable or superior antiproliferative activity compared to the clinically prescribed 1 and irinotecan (3). Significantly, the 20-sulfonylamidine derivatives exhibited comparable cytotoxicity against KBvin, while 1 and 3 were less active against this cell line. Among them, compound 15c displayed much better cytotoxic activity than the controls 1, 3, and 9. Novel key structural features related to the antiproliferative activities were identified by structure–activity relationship (SAR) analysis. In a molecular docking model, compounds 9 and 15c interacted with Topo I-DNA through a different binding mode from 1 and 3. The sulfonylamidine side chains of 9 and 15c could likely form direct hydrogen bonds with Topo I, while hydrophobic interaction with Topo I and π–π stacking with double strand DNA were also confirmed as binding driving forces. The results from docking models were consistent with the SAR conclusions. The introduction of bulky substituents at the 20-position contributed to the altered binding mode of the compound by allowing them to form new interactions with Topo I residues. The information obtained in this study will be helpful for the design of new derivatives of 1 with most promising anticancer activity.CPT (green), 9 (magenta), and 15c (blue) in the binding site of DNA-Topo-I