C This famiiarlae queition has several parts and includes several of the molecul
ID: 187583 • Letter: C
Question
C This famiiarlae queition has several parts and includes several of the molecular techniques that we learned about. First read the abstract to e yourself with the research topic, and then go on to answer the questions about the data shown below ABSTRACT (modified from Chan et al. Journal of Immunology 2005) A marked difference exists in the refers inducibility of the gene inducible NO synthase (GNOS) between humans and rodents. (Inducibility hanged.) The rodent iNOS gene can be induced to be expressed at high pes of eytokines (a type of protein generated by immune cells), but these same cytokines have little to no effect on s gene. A compelling molecular explanation for why human iNOS is resistant to induction has not byporesponsiveness of the human iNOS promoter is based in part on to the degree to which the expression of a gene can be c expression of the human iNO been reported. epigenetic silencing. In this study we present evidence that the Part I (3 points): The researchers first need to establish the system that they will use in their paper. The cell type they will use for their experiments is human umbilieal vein endothelial cells (HUVEC), Based on the data in Figure IA, what can you conclude about the inducibility of the iNOS gene and the VCAM-1 gene in HUVEC cells in response to cytokine stimulation? Explain your reasoning. Part II (3 points): What can you conclude about the data in Figure 18? How do the data in panel B support your conclusion in Part 1? Real-Time RT-PCR Pol lychiP VCAM-131 INOs 12 on iNos and VCAM-1 steady-state mRNA levels and transcription HUVEC. Primary HUVEC were ml), and Data represent the mean ± SEM of four independent experiments. B.CMP assay using an antibody directed against RNA polymerase II (Pol ID. IP DNA was analyzed by quantitative PCR using iNOs and VCAMI promoter-specific primers. Data represent the imcan a stimulated with vehicle (negative control), TNF-a (10 ng'ml), or a cytokine mixture (Cyt) of IFN-y (200 U/ml), ILIB (5 ngl INF a (10 ng/ml) for 4 h, after which RNA and chromatin extracts were i isolated. A. Quantitative RT-PCR of human iNOS and CAM-I mRNA. Inducible NOS and VCAM-1 mRNA expressions were normalized to GAPDH mRNA level (in arbitrary units) SD of triplicate measurements from one of four independent esperiments with similar results. .pExplanation / Answer
ANS. Expression of iNOS is quantified by the quantitative riverse transcriptase - polymerase chain reaction.the DNA methylation status of the iNOS promoter and enhancer regions was determined by the busulfite sequencing or pyrosequencing. the effect of CpG methylation on iNOS promoter and enhancer activities was determined using a CpG luciferase vector and a CpG methyltransferase. cotranfections with expression vector encoding NF-kB subunits were carried out to analyze iNOS promoter and enhancer activites in response to changes in methylation status.
The iNOS promoter construct (piNOS)and the iNOS enhancer plus promoter construct(epiNOS) were methylated in vitro using CpG methyltransferase to analyze the affect of CpG methylation on iNOS activity.the suppressive effect of CpG methylation on iNOS proximal promoter was not significant.similar results were observed when subunits of NF-kB over expressed by cotransfection.in contrast,with the epiONS construct,the enhanced iNOS activity induced by p65 overexpression was significantly reduced after methylation treatment.this supressive effect of methylation also occured with the p65/p50 driven transactivated epiONS.
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.