# of band visible on the gel assuming all unprotected DNA was cleaved? Mast.Gen.

A. We will see the DNA fragments of sizes 145-200 bps in the gel present in one single band.


B. Since we are removing the non histone proteins first, only the DNA associated with histone proteins will be protected from the action of DNase I. 145 bp of DNA is attached with histone proteins and each nucleosome is attached with each other by linker DNA. DNase I might cut at any portion of 55 bp linker DNA. So, we are expected to see fragments of 145bp to 200 bp nucleotides in the gel. But since the difference between the bands won't be significant we will see a single band in the 145-200 bp region.

a) As the non-histone proteins have been removed from the DNA sequence, the DNase 1 enzyme will have access to the portions of DNA except the portions which have the nucleosomes. Thus, the 145 bp fragments will remain intact. However the linker DNA (55 bp) is free of proteins and thus can be cleaved by the DNase1 enzyme. DNase 1 cleaves specifically next to pyrimidine nucleotides on the DNA and can produce fragments of DNA ranging from 4 bp to above. But generally, it gives tetranucleotides.

Thus, prominently 2 bands shall be seen on the gel. 1 band represents the 145 bp fragments that have not been affected by DNase1. The other band will represent the fragments that are of very small size i.e 4-50 bp. As these small fragments can not be effectively determined and differentiated in an agarose gel, they will hence give one uniform band.

b) The origin of the fragments seen in the gel as stated above is from the DNA wrapped around the nucleosomes (145 bp) and the linker DNA (55 bp). The band that travels further represents the band with fragments ranging from >= 4bp, and these fragments are formed due to DNase activity on the protein free linker DNA. Whereas the band that is closer to the wells represents the 145 bp fragments which were wrapped around the nucleosomes and have not been cleaved or affected by the DNase as they still had histone proteins.

Answer is 1 band