2 . Nuclei have been isolated from a stem cell line at 0, 1, 2, and 4 days after

Question

2.         Nuclei have been isolated from a stem cell line at 0, 1, 2, and 4 days after the cell line has been induced to differentiate into muscle type cells. The nuclei are subjected to digestion with DNase, the DNA is then extracted from the nuclei and cleaved with a restriction enzyme that cleaves the DNA external to genes X and Y. The digestion products are run on a gel and analyzed by Southern blots using a labeled probe that binds to either X or Y DNA (or RNA). The results of this experiment are shown on the Southern blot below at the left. The experiment is repeated, but without DNase, and the results are shown on the Southern blot below in the middle. Finally, RNA, extracted from the cells at days 0, 1, 2 and 4 after induction, is run on gels which are then blotted and probed for the presence of X or Y RNA, as shown in the Northern blot below, right.

a.         Why do the patterns of bands differ between the two Southern blots?

b.         What is a likely explanation why the pattern of bands on the Southern blot at the left differs from the pattern on the Northern blot?  

c.         Is either gene X or gene Y likely to encode a muscle cell specific protein?

d.         Is either gene X or gene Y likely to encode a “housekeeping” protein? (Housekeeping proteins are required for the survival of all cells.)

e.         If the histone acetylase activity in the stem cell line above were to be inhibited, how would the results obtained on any of the three blots above likely differ?

Explanation / Answer

a) because treatment of DNase reveals DNase hypersensitive sites. A stretch of DNA gets hypersensitive due to DNA if it is free from proteins and open for transcription i.e. it is acetylated. When DNase treatment is not given all bands seem similar.

b) intact band in the left southern blot indicates that it is transcriptionally inactive since it is not sensitive to DNase hence it will not form RNA. Vice versa holds true.

c)gene y seems to be muscle specific as it's expression peaks as the stem cell gets more and more dedicated to the muscle cell type.

d. no. Housekeeping genes are needed always at the same amount. X and y gene expression is not consistent with time.

e) the 2 southern blots would be identical as there would be not DNase hypersensitive site. The northern blot would show 0 or low level expression of both genes as both are not accessible to the transcription machinery by histone acetylase.

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