7(4pts). You join a summer research program in a lab that is trying to understan

Question

7(4pts). You join a summer research program in a lab that is trying to understand cells response to cold. You grow half the cells at room temperature (23°C) and the other half at 15°C After two hours incubation, you isolate the DNA from cells using a gentle procedure that leaves nucleosomes and some higher order chromatin structures intact. You then treat the DNA for a short time with a low concentration of M-nuclease, an enzyme that degrades protein-free stretches of DNA. After removing proteins, you separate the resulting DNA on the basis of length using gel electrophoresis. Finally, you visualize the DNA fragments from a region near the Brr1 gene or the Brr2 gene shown on the gel below. Remember that smaller DNA molecules move faster through a gel and are found near the bottom of the gel. Darker spots contain more DNA than fainter spots The lanes are as follows: lane "marker" containing known DNA fragments of indicated lengths 2. Cells grown at 23°C, visualized DNA near Brr1 gene 3 4. 5. Cells grown at 15°C, visualized DNA near Brr2 gene Cells grown at 15°C, visualized DNA near Brr1 gene Cells grown at 23°C, visualized DNA near Brr2 gene A. There is a fragment of DNA at 150 nucleotides in each lane 2-5. What would this smallest fragment of DNA correspond to and why is it 150 nucleotides in length? B. Lane 2 has a ladder of spots with longer lengths. Why are the spots evenly spaced? What do they correspond to? C. Notice the faint spots and extensive smearing in lane 3, suggesting the DNA could be cut almost anywhere near the Brr1 gene after growth of the cells at 15°C. This was not observed in the other lanes. What probably happened to the DNA to change the pattern between lanes 2 and 3? D. Describe the relative expression levels of Brr1 and Brr2 at 15°C and 23°C. Where is gene expression high and low?

Explanation / Answer

Eukaryotic DNA is packaged into extremely compact structures, the structural units of which are called nucleosomes. Such a tight packaging, in addition to packing, determines the accessibility of the gene expression machinery to the genes present in the nucleosomes, an area of research called epigenetics. A nucleosome consists of histone proteins, around which 146bp of the DNA is wrapped, making a 'beaded' appearance. Each such bead is linked to its adjacent one by 20-60bp linker DNA. Thus, A DNAse treatment cannot access the DNA wrapped around the nucleosomes. Moreover, a partial digestion of the chromosome will yield DNA fragments having sizes of multiple of 186 (approx., 146bp + 40 = 186bp).

A. The 150bp band is the size of DNA wrapped in the nucleosomes, the smallest length inaccessible to the nuclease treatment.

B) The nuclease treatment was partial (low concentration of M-nuclease, as mentioned in the question). Therefore, it has missed some of the linker DNA sites and therefore, sizes of multiples of 186 can be seen (for example, 2x186=372, 3x186=558 and so on).

C) At 15 deg C, nucleosomes near the Brr1 gene were unfolded, allowing access to the M-nuclease. This also means that the Brr1 gene gets expressed at 15deg, but not at room temperature.

D) Brr1 is expressed at 15deg, but not at 23deg. Brr2 gene is not expressed in any of the two temperatures. Therefore, Brr1 gene expression will be higher at 15deg C.

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