A. [4pts] First, you examine the chromosomal DNA. You isolate DNA from cells, la

Question

A. [4pts] First, you examine the chromosomal DNA. You isolate DNA from cells, label 5' ends with radioactive ATPby using polynucleotide kinase, run the DNA on a gel, and detect radiolabeled fragments. Your results are shownin Figure A . The lane from the untreated control cells is labeled U. Does X cause apoptosis? What about Y? Explainthe origin of the ladder in the Y lane.

B. [4pts] Second, you examine the subcellular localization of cytochrome c using immunofluorescencemicroscopy. Unfortunately, during sample preparation you mixed up the samples. Assign a likely identity (U, X, orY) to the samples labeled 1, 2, and 3 in Figure B.

C. [4pts] Third, without making the cells permeable in any way, you stain them with Annexin V linked to a greenfluorophore, which binds phosphatidylserine, and propidium iodide, which fluoresces red when it binds DNA.Neither of these compounds can travel across intact plasma membranes. Again during sample preparation youmixed up the samples. Assign a likely identity to the samples in Figure C. Annexin V staining is shown as \hatching, propidium iodide staining is shown as // hatching, and staining with both in the same region appears ascrosshatching

Explanation / Answer

A. The drug Y causes apoptosis, as evident from the DNA laddering pattern that can be observed here. DNA laddering is a result of degradation of the DNA and these fragments appear as aggregates of different multiples. B. Cytochrome c release into cytosol shows the occurance of a possible apoptosis. Both 1 and 3 show bright speckles of cytochrome c in the cytoplasm and therefore 1 and 3 could be Y and 2 is U for sure since first figure shows no apoptosis. C. Better staining of Annexin and PI is required here to assess the results. But for your analysis Annexin enters into cells: indicates apoptosis. PI enters the nucleus: indicates necrosis Entry of both: Apoptosis followed by necrosis.

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