Question
Consider Figure 12.22a. What primers would you use in a 3C experiment to show association between the ICR insulator and each of the $I g f 2$ promoters $P 1, P 2$, and $P 3$, on the maternal chromosome.
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In this case, we need to focus on the ICR insulator and the three Igf2 promoters (P1, P2, and P3) on the maternal chromosome. Show more…
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The image below shows the general structure of a gene on a chromosome. The arrows above and below the chromosome indicate the binding positions of potential forward (F) and reverse (R) PCR primers. Select two primers from the list below that would exclusively amplify exon 3 in a PCR reaction. Intron 1 Intron 2 Poly-A signal ATG TAG F6 Exon 3 R1 F2 R5 F3 F4 F5 Exon 1 Exon 2 R6 R4 R3 R2 Promoter Select all that apply: a. F1 b. F2 c. F3 d. F4 e. F5 f. F6 g. R1 h. R2 i. R3 j. R4 k. R5 l. R6
HP1 proteins, a family of proteins found in heterochromatin, are implicated in gene silencing and chromatin structure. The three proteins in humans- HP1 $\alpha, \mathrm{HP} 1 \beta,$ and HP1 $\gamma$ -share a highly conserved chromodomain, which is thought to direct chromatin localization. To determine whether these proteins could bind to the histone H3 N-terminus, you have covalently attached to separate beads various versions of the H3 N-terminal peptide -unmodified, Lys-9-dimethylated (K9-Me), and Ser-10-phosphorylated $(\mathrm{S} 10-\mathrm{P})-$ along with an unmodified tail from histone $\mathrm{H} 4$. This arrangement allows you to incubate the beads with various proteins, wash away unbound proteins, and then elute bound proteins for assay by Western blotting. The results of your 'pull-down' assay for the HP1 proteins are shown in Figure $Q 4-3,$ along with the results from several control proteins, including Pax5, a gene regulatory protein, polycomb protein Pcl, which is known to bind to histones, and Suv39h1, a histone methyltransferase. Based on these results, which of the proteins tested bind to the unmodified tails of histones? Do any of the HP1 proteins and control proteins selectively bind to the modified histone N-terminal peptides? What histone modification would you predict would be found in heterochromatin?
Two wild-type fragments of human genomic DNA from the long arms of two nonhomologous acrocentric chromosomes are shown in the accompanying diagram. On both fragments, the centromere-to-telomere direction is left-to-right. The red lines numbered $1,2,$ and 3 indicate the positions of breakpoints that could be caused by ionizing radiation. Certain combinations of these breaks could produce chromosomal rearrangements when the broken pieces are stitched back together by DNA repair systems. You will want to diagnose the presence of the rearrangements by using PCR. In every case that follows, one of the primers you will use for PCR is primer $A,$ whose sequence is: 5' TCGATTCCGGAAAGCT 3' a. Which two of the breakpoints could yield a deletion? b. Write the sequence and polarity of a 16 -base primer that, in conjunction with primer $A,$ will allow you to diagnose the presence of a deletion in a patient's genomic DNA. The evidence should be positive, not negative. (That is, you will see a PCR product whose presence and/or size is specific to the deletion; the absence of a band is not informative.) c. Which two of the breakpoints could yield an inversion? Write the sequence and polarity of a 16 -base primer that, in conjunction with primer $A,$ will allow you to diagnose the presence of an inversion in a patient's genomic DNA. Your answer should show the primer that would yield the longest possible diagnostic PCR product. Your primer cannot cross any of the red lines. e. Which two of the breakpoints could yield a reciprocal translocation? (Two possible answers exist; you need only write one.) f. Write the sequence and polarity of a 16 -base primer that, in conjunction with primer A, will allow you to diagnose by PCR the presence of a reciprocal translocation in a patient's genomic DNA. This reciprocal translocation can be stably inherited through meiosis without its loss or any chromosomal breakage. Again, the evidence should be positive, not negative. g. Is the translocation you described in parts (e) and (f) Robertsonian? Answer yes or $n o,$ and explain.
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