ABSTRACT (modified from Chan et al. Journal of Immunology 2005) A marked differe

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ABSTRACT (modified from Chan et al. Journal of Immunology 2005) A marked difference exists in the inducibility of the gene inducible NO synthase (INOS) between humans and rodents. (Inducibility refers to the degree to which the expression of a gene can be changed.) The rodent iNOS gene can be induced to be expressed at high levels by several types of cytokines (a type of protein generated by immune cells), but these same cytokines have little to no effect on expression of the human iNOS gene. A compelling molecular explanation for why human iNOS is resistant to induction has not yporesponsiveness of the human iNOS promoter is based in part on reported. In this study we present evidence that the h epigenetic silencing. Part I (3 points): The researchers first need to establish the system that they will use in their paper. The cell type they vwill use for 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 umbilical vein endothelial cells (HUVEC), Based on the data in Figure 1A, what can you conclude about ty of the INOS gene and the VCAM-18 at can you conclude about the data in Figure 18? How do the data in panel 8 support your conclusion in Part ? B. Real-Time RT-PCR Pol IVChIP 16 VCAM-1 CAM-1 12 n es on iNOS and VCAM-1 steady-state mRNA levels and transcription HUVEC. Primary HUVEC were with vehicle (negative control), TNF- (10 ng/ml), or a cytokine mixture (Cyt) of IFN-Y (200 U/nl), IL. 1 (5 ng/ml), and TNF a (10 ng/ml) for 4 h, after which RNA and chromatin extracts were isolated. A.Quantitative RT-PCR of human iNOS and VCAM-1 mRNA. Inducible NOS and VCAM-1 mRNA expressions were normalized to GAPDH mRNA level (in arbitrary units) Data represent the mean ± SEM of four independent experiments. B ChIP assay using an antibody directed against RNA polymer Il (Pol il). IP DNA was analyzed by quantitative PCR using iNOS and VCAM/ promoter-specifie primers. Data represent the mean t SD of triplicate measurements from one of four independent experiments with similar results..,p

Explanation / Answer

1. The major differences between the human and the murine iNOS activation based on the promoter activity.
For murine promoter 1.5 kb of the 5 flanking region is enough to activate by LPS and interferon-gamma (IFN-),
But in the human iNOS promoter is hyporesponsive to LPS/IFN- due to nucleotide exchanges in the LPS/IFN--responsive enhancer region.

The human transcription factor NF-B, induced by treatment with IL-1, tumour necrosis factor-alpha (TNF-) and IFN-, binds to the iNOS promoter more weakly than mouse NF-B.

Thats why there is no expression of mRNA in the iNOS gene compare to the VCAM-1.

2. ChIP-seq is used primarily to determine how transcription factors interact with DNA to regulate gene expression.
We already seen that human transcription factor are weakly binds to the DNA. That why the iNOS bar graph shows little expression compare to the VCAM 1.

3. It clearly shows that Histone H3 lysine 9 methylation opens the promoter activity for cytokines. So, cytokine binds to the DNA act as a transcription factor and induces the expression INOS mRNA. So, in Ip DNA there is increase in activity. But same time VCAM-1 is arrested so there is no expression.

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