End of of unit questions
What effect does ethidium bromide intercalation have on supercoiled DNA?
When ethidium bromide intercalates into duplex DNA, it results in an unwinding of the helix through disruption of the base-stacking interactions that contribute to its structure. This untwisting of the helix reduces the energy available for supercoiling. Hence, supercoiled DNA, when incubated with ethidium bromide, becomes less supercoiled.
Imagine that you have recently isolated from a gene library a segment of human DNA that corresponds to a coding region. How could you investigate in the laboratory the chromosomal location of this gene and the sites of its expression?
The chromosomal location of the gene could be determined by FISH, where the DNA is labelled and hybridized to metaphase chromosomes. The position of the fluorescent signal on the chromosome should indicate the position of the gene. The expression pattern can be determined in one of two ways. Firstly, the DNA can be used to perform ISH on tissue sections. Secondly, it could be used as a probe against a Northern blot of mRNA taken from various human tissues.
What three roles does DNA packaging fulfil within the cell?
Chromatin packaging serves (1) to compact the large amounts of DNA in a small space, (2) to protect DNA from damage, and (3) as a platform for DNA processing.
Why does the deacetylation of histone tails allow closer packing of nucleosomes?
Histone tails are rich in positively charged lysine residues, many of which are subject to modification by acetylation (and also phosphorylation and methylation). Removal of acetyl groups from the tails exposes the positive charge on the tails, which in turn allows the tails to counteract the repulsion of negatively charged DNA backbones, hence permitting closer packing of the DNA.
What effect does the wrapping of DNA around the histone octamer in the nucleosome have upon the DNA?
Two left-handed super-helical turns are introduced to the DNA, so that it adopts a solenoid-like structure around the core.
Describe the major modifications that occur to core nucleosomal proteins. How do these modifications exert their effects?
Modifications of core nucleosomal proteins include covalent modification of the tails of core histones by acetylation, methylation or phosphorylation. Acetylation or methylation of lysine residues in the histone tails reduces the net positive charge associated with this part of the protein and affects its ability to neutralise the repulsive forces between regions of the DNA backbone. As a result, compaction of the DNA is decreased. Modification of histones can also affect chromatin compaction and transcriptional activity by recruiting regulatory proteins to specific regions.