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Biology 463: Techniques Presentation Question
Depiction of the -globin locus in mice, which is roughly 200 kb in length
- The black arrows correspond to the  and h1 genes, which are inactive in fetal
brain and liver cells.
- The red arrows correspond to the maj and min genes, which are active in fetal
liver cells, but inactive in fetal brain cells.
- The shaded regions containing a given roman numeral are restriction fragments.
- The red dots correspond to DNaseI hypersensitivity (HS) sites. HS sites 1-6
represent the -globin locus control region (LCR), which is known to enhance the
expression of active -globin genes.
- Relative cross-linking frequencies observed in fetal liver cells are shown in red,
while those in fetal brain cells are shown in blue.
- The relative cross-linking frequency is (roughly speaking) the amount of crosslinked fragments measured by quantitative PCR (qPCR) in the cell nuclei
compared to the amount of ligated fragments measured by qPCR in a
standardized control with equimolar amounts of each possible ligated fragment.
- Each data point in diagram A represents the relative cross-linking frequency of
the given restriction fragment with fragment VIII (which contains the maj gene).
- Each data point in diagram C represents the relative cross-linking frequency of
the given restriction fragment with fragment VII (which contains the h1 gene).
With all of the relevant information above, decide which of the following statements
are true.
1) If we wanted to examine the interaction of the HS sites in the LCR with the ßglobin genes more precisely, we would need to use restriction enzymes that cut
this section of the ß-globin locus more frequently.
2) If relative cross-linking frequencies are roughly monotone decreasing as we
move from adjacent fragments on the chromosome to more distant fragments on
the chromosome, this suggests a linear chromosome conformation. For this
reason, the -globin locus in brain cells appears to have a roughly linear
conformation.
3) The LCR (fragments IV-VI), a known enhancer, is in closer spatial proximity to
active -globin genes than inactive -globin genes.
4) If fragments II and IV had relative cross-linking frequencies greater than 1, this
would imply that they were in closer spatial proximity to one another compared
to if the -globin locus were linear in conformation.
5) The fact that fragment III has similar relative cross-linking frequencies in both
fetal liver and brain cells with both inactive and active -globin genes, implies
that it is not in close spatial proximity to such genes via loop formation.
Answer: Only statements 1-3 are true.
Explanation:
1) This statement is true because if the restriction enzymes cut more frequently, it is
more likely that each individual HS site in the LCR will belong to its own
restriction fragment. Then, the relative cross-linking frequency of each HS site
with the active -globin genes can be determined, allowing for a more detailed
mapping of the conformation of the -globin locus.
2) This statement is true because in a linear chromosome, fragments that are
adjacent on the chromosome are also adjacent in space, and the farther two
fragments become on the chromosome, the farther apart in space they become.
Thus, relative cross-linking frequencies will be monotone decreasing as
fragments become farther apart on the chromosome, which is roughly what we
see in the fetal brain cells, especially when compared to the fetal liver cells.
3) This statement is true because the relative cross-linking frequencies are much
higher between the LCR and the active -globin gene (namely fragment VIII in
fetal liver cells) compared to the relative cross-linking frequencies between the
LCR and the inactive -globin genes (fragment VIII in fetal brain cells and
fragment VII in both fetal brain and liver cells).
4) This statement is false because a relative cross-linking frequency of 1 is an
arbitrary number, having nothing to do with the relative cross-linking frequency
of two fragments on a linear chromosomal segment. If, however, fragments II and
IV had relatively high cross-linking frequencies compared to, say, fragments I and
IV, fragments III and IV, and fragments II and III, we could conclude that
fragments II and IV were in relatively close spatial proximity compared to other
nearby fragments, suggesting a loop formation between the two.
5) This statement is false because if the relative cross-linking frequencies in these
situations were all relatively high compared to the cross-linking frequencies of
other nearby fragments, this would suggest that a loop formation occurs in all
four situations, independent of gene activation. Evidently, what is important is
the relative cross-linking frequencies of two fragments compared to other nearby
fragments that determines whether they are in close spatial proximity to one
another. So, for example, the fact that fragments IV, V and VI have higher relative
cross-linking frequencies with fragment VIII, compared to both fragment VII and
fragment III in fetal liver cells, suggests that a loop formation occurs between the
LCR and the active gene. Comparatively, in the fetal brain cells, fragments IV, V
and VI have much lower relative cross-linking frequencies with fragment VIII
compared to fragment VII, which is more consistent with a linear chromosome
conformation.. The key is that while it is true that fragment III is not in close
spatial proximity to the -globin genes, the implication is false because the
predicate does not guarantee that this statement will be true.