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

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 Brrl 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 Cells grown at 23°C, visualized DNA near Brrl gene Cells grown at 15°C, visualized DNA near Brrl gene Cells grown at 23°C, visualized DNA near Brr2 gene Cells grown at 15°C, visualized DNA near Brr2 gene 4 1000 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? 500 150 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 Brrl 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 Brri and Brr2 at 15°C and 23°C. Where is gene expression high and low?

Explanation / Answer

Ans. (A). There is a fragment of DNA at 150 nucleotides in each lane 2-5. This smallest fragment of DNA corresponding to the actual size of the Brr1 and Brr2 gene that is 150 base pairs. Therefore, at 150 base pair size, the fragment is fully resolved and separated.

(B). The lane 2 has ladder spots with the longer length. Each spot is evenly spaced because in gel electrophoresis the molecules get separated on the basis of the molecular weight. The molecular weight is inversely proportional to the migration rate through a gel matrix. So each band corresponding to the respective size of the fragments.

(C). The DNA pattern changes drastically between lane 2 and 3. This is so because may be genomic DNA gets degraded in the lane 3 or maybe the loading error or somehow forget to add DNase inhibitors during the extraction process. That is why faint spots and extensive smearing is observed in lane 3.

(D). The relative expression levels of Brr2 gene found higher as compared to the Brr1 gene at 15oC. While the expression of Brr1 gene and Brr2 gene found to be more or less equal.

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