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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.