Question

can someone explain how to answer this with reasons why

30-45 mins after estrogen addition: Acchyiase (HAT, odde auhye 60-90 mins after estrogen addition: Reauitment ef Poy to open 7. T identify the Gene X DNA element responsible for regulation by a Growth Fagor, you perform a linker scanning experiment in which you mutate 10 bp Tegions centered around positions -205 to-5 in the Gene X promoter/promoter- proximal region (wt wild-type promoter). The promoter variants were tested for their ability to activate transcription of Gene X in the absence (-) or presence (+)of the Growth factor. mportant Mutated region: -205 -155 -125 -8565 45 35 25 -15 wt -Growth factor mRNA from Gene + Growth factor mRNA from Gene (a) Based on the +Growth factor experiment, list which regions contain sequences that Mutated region: -205 -155 -125 -85 -65 -35 -25 -15 -45 wt - Growth factor mRNA from Gene X +Growth factor mRNA from Gene X (a) Based on the '+ Growth factor experiment, list which regions contain sequences that, -C, -s Stimulate transcription of Gene X: - 1SS,-n5, Repress transcription of Gene X:&S (b) Treatment of cells with a Growth factor causes a proto-oncogene transcription activator called ONC to activate Gene X. Given that ONC only activates transcription when the Growth factor is present, which region is most likely to contain the ONC binding site (note: there is only one). (c) What is the name of the blot shown at the top of this page? Next, you treat cells with or without Growth hormone, isolate total protein from the nucleus (N) or cytoplasm (C) of the cells and perform an electrophoretic mobility shift assay (EMSA) using a s2P-labeled DNA containing the ONC binding site. (The control in lane 1 shows the binding assay performed without protein). Growth hormone: Nuclear (N) or cytoplasmic (C):. N CN C (d) In which lane(s) (1,2,3,4 and/or 5) of the shown EMSA does ONC bind the DNA? Edit Next, you treat cells with or without Growth hormone, isolate total protein from the nucleus (N) or cytoplasm (C) of the cells and perform an electrophoretic mobility shift assay (EMSA) using a s2P-labeled DNA containing the ONC binding site. (The control in lane 1 shows the binding assay performed without protein). Growth hormone:. Noelear (N) er eytoplasmie (CE (d) In which lane(s) (1,2,3,4 and/or 5) of the shown EMSA does ONC bind the DNA? NC Lane: 1 2 345 (e) Based on this result, what is the simplest hypothesis for why ONC only activates Gene X transcription in the presence of the Growth hormone. 8. Youperform chromatin immunoprecipitation (ChIP) assays on cells not treated (-) or treated (+) with the Growth factor, using antibodies specific for the following factors: Growth factor: i) ONC i) TFIID (the factor that binds the TATA-box) i) RNA polymerase II v) Phospho-CTD (The phosphorylated form of RNA Pol-ll C-terminal domain) v) Histone H4. ONC TFIID RNA pol I phospho-CTD Your ChiP assays are analyzed by PCR for the promoter and an enhancer region of Gene X. Histone H4 ChIP assays (a) Based on the result of the ChiP assay, on the schematics below draw and clearly label what is present at the Gene X promoter/promoter-proximal region in the absence and presence of the Growth factor. (No need to indicate the specific positions of factors). PP/P gene - Growth factor:

Answer 1

Explanation:-

1. Linker scanning experiment used to introduce mutation in desired promoter or regulatory segment in the form of point mutation. This leads to loss of function and helps to identify the role of the particular sequence in which mutation has occurred.
2. In the above mentioned sequence, mutation has been introduced in -5 to -205 positions.
3. When the mRNA from gene treated with growth factor is observed, the expression levels can be seen in the form of bands. Higher expression indicates over activity and small bands indicates reduced expression more specifically due to mutation.
4. When treated with growth factor, all other regions were equally expressed except -155, -125, -45 and -25 to show under expression and thus may lead to initiation of the function if present in optimum level.
5. Band positioned -85 is over expressed indicating its role in negative regulation as is available to express during addition of growth factor.