You isolate chromatin from mouse liver cells (hepatocytes). You set aside one sa

Question

You isolate chromatin from mouse liver cells (hepatocytes).

You set aside one sample and isolate DNA from it – removing all traces of protein.

You treat another 5 samples (equal amounts) of the chromatin with increasing amounts of DNAseI enzyme.

After you inactivate the DNAseI in these samples, you isolate the DNA from each (again, removing all traces of protein).

Now you cut all 6 of your isolated DNA samples with the restriction endonuclease EcoRI.

You run all 6 samples on an agarose gel (to separate the DNA fragments according to size).

You do a Southern Blot, and hybridize the nylon sheet with a DNA probe that you know is homologous to a region near an EcoRI site.

These are your results:

Part A -

Mark any answer below that is NOT demonstrated by these results. (THERE CAN BE MORE THAN ONE ANSWER)a)

a) There is a DNAseI hypersensitive site 2kb away from one end of the 9kb EcoRI fragment

b) There is likely a regulatory region (enhancer/promoter/silencer) 2kb away from one end of the 9kb EcoRI fragment

c) In the mouse genome, there is a 9kb EcoRI DNA fragment that is homologous to your probe

d) There is a DNAseI hypersensitive site 7kb away from one end of the 9kb EcoRI fragment

e) In the mouse genome, there is a 2kb EcoRI DNA fragment that is homologous to your probe

a) There is a DNAseI hypersensitive site 2kb away from one end of the 9kb EcoRI fragment

b) There is likely a regulatory region (enhancer/promoter/silencer) 2kb away from one end of the 9kb EcoRI fragment

c) In the mouse genome, there is a 9kb EcoRI DNA fragment that is homologous to your probe

d) There is a DNAseI hypersensitive site 7kb away from one end of the 9kb EcoRI fragment

e) In the mouse genome, there is a 2kb EcoRI DNA fragment that is homologous to your probe

Explanation / Answer

Answer: Option e) is NOT demonstrated by given results.

Explaination: Deoxyribonuclease I (DNaseI), is an endonuclease tend to cleaves DNA at phosphodiester linkages near to a pyrimidine nucleotide (C, T). It can cleave single-stranded DNA, double-stranded DNA, and chromatin as well.

When given 5 samples of mouse chromatin was treated with DNaseI, it cleave the mouse genome into DNA fragments. However, the control genome sample (No DNaseI) not treated with DNaseI, not cleaved into fragments.

When all the 6 DNA samples are treated with EcoRI (restriction endonucleases cut at the recognition sequence G/AATTC), and after that run on an agarose gel (to separate the DNA fragments according to size), the 9kb band is appeared in the southern blot, hybridize with a DNA probe (homologous to a region near an EcoRI site). This result of single 9kb band of control genome sample, demonstrated that in the mouse genome, there is a 9kb EcoRI DNA fragment that is homologous to your probe.

DNAseI hypersensitive sites are found in regulatory regions where nucleosomes have been removed or moved to allow regulatory proteins (e.g. transcription factors) to bind the DNA. Thus, there is likely a regulatory region (enhancer/promoter/silencer) 2kb away from one end of the 9kb EcoRI fragment.

However, in the rest of 5 lanes of southern blot, two band are appeared (i.e.of 2kb and 9kb), as the genome was cleaved by the DNaseI. Therefore, DNAse treatment is causing the 9kb EcoRI fragment to be cut into at least two pieces. It is known that the probe is near one end of the EcoRI fragment, the conclusion is that there is a DNAse hypersensitive site 2kb away from that end of the EcoRI fragment and 7kb away from another end of the EcoRI fragment.

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