CHAPTER 17
Questions for Student Discussion/Collaboration
1.
Explain how DNA methylation could be used to regulate gene expression in a
tissue-specific way. When and where would de novo methylation occur, and when would
demethylation occur? What would occur in the germ line?
Answer: DNA methylation can regulate gene expression in a tissue-specific way because
de novo methylation may occur only next to particular genes in particular tissues. This
would tend to keep them permanently turned off during later stages of development.
Genes may be methylated in the germ line to prevent their expression. However, if a gene
was methylated in the germ line, it would have to be demethylated during gamete
formation or at an early stage of embryonic development in the offspring so that it could
be expressed in particular tissues in the offspring. Alternatively, genes that are regulated
in a tissue-specific way may not be methylated in the germline, but instead are
methylated during embryonic development in tissues where their expression should be
silenced.
2.
Enhancers can be almost anywhere and affect the transcription of a gene. Let’s
suppose you have a gene cloned on a piece of DNA, and the DNA fragment is 50,000 bp
in length. Using cloning methods described in Chapter 19, you can cut out short segments
from this 50,000 bp fragment and then reintroduce the smaller fragments into a cell that
can express the gene. You would like to know if any enhancers are within the 50,000 bp
region that may affect the expression of the gene. Discuss the most efficient strategy you
can think of to trim your 50,000 bp fragment and thereby locate enhancers. You can
assume that the coding sequence of the gene is in the center of the 50,000 bp fragment
and that you can trim the 50,000 bp fragment into any size piece you want using
molecular techniques described in Chapter 19.
Answer: Probably the most efficient method would be to systematically make deletions
of progressively smaller sizes. For example, you could begin by deleting 20,000 bp on
either side of the gene and see if that affects transcription. If you found that only the
deletion on the 5' end of the gene had an effect, you could then start making deletions
from the 5' end, perhaps in 10,000 bp or 5,000 bp increments until you localized
regulatory elements. You would then make smaller deletions in the putative region until it
was down to a hundred or a few dozen nucleotides. At this point, you might conduct sitedirected mutagenesis as described in Chapter 19, as a way to specifically identify the
regulatory element sequence.
3.
How are regulatory transcription factors and regulatory splicing factors similar in
their mechanism of action? In your discussion, consider the domain structures of both
types of proteins. How are they different?
Answer: Transcription factors and regulatory splicing factors are similar in that they
recognize a particular nucleotide sequence. They can act in a tissue-specific or
developmentally specific manner. Likewise, they are similar in that they influence the
activity of some other multisubunit complex. Regulatory transcription factors affect the
activity of RNA polymerase and general transcription factors; splicing factors affect the
splice sites of the spliceosome. Regulatory transcription factors and splicing factors have
multiple domains that play different functional roles; they have modular structures. For
example, regulatory transcription factors have domains for nucleotide binding and may
have domains for dimerization, transactivation, and so forth. Regulatory transcription
factors and splicing factors are different in that the former recognizes DNA, the latter
RNA. The role of regulatory transcription factors is to influence the rate of transcription,
whereas the role of splicing factors is to affect the specificity of splice-site recognition.