Ch. 12 - DNA Organization in Chromosomes
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Problem 1
In this chapter, we focused on how DNA is organized at the chromosomal level. Along the way, we found many opportunities to consider the methods and reasoning by which much of this information was acquired. From the explanations given in the chapter, what answers would you propose to the following fundamental questions: How do we know that viral and bacterial chromosomes most often consist of circular DNA molecules devoid of protein?Problem 1
In this chapter, we focused on how DNA is organized at the chromosomal level. Along the way, we found many opportunities to consider the methods and reasoning by which much of this information was acquired. From the explanations given in the chapter, what answers would you propose to the following fundamental questions: How did we learn that eukaryotic chromatin exists in the form of repeating nucleosomes, each consisting of about 200 base pairs and an octamer of histones?Problem 2
Write a short essay that contrasts the major differences between the organization of DNA in viruses and bacteria versus eukaryotes.Problem 3
Contrast the size of the single chromosome in bacteriophage and T2 with that of E. coli. How does this relate to the relative size and complexity of phages and bacteria?Problem 4
Describe the structure of giant polytene chromosomes and how they arise.Problem 5
What genetic process is occurring in a puff of a polytene chromosome? How do we know this experimentally?Problem 6
During what genetic process are lampbrush chromosomes present in vertebrates?Problem 7
Why might we predict that the organization of eukaryotic genetic material will be more complex than that of viruses or bacteria?Problem 8
Describe the sequence of research findings that led to the development of the model of chromatin structure.Problem 9
Describe the molecular composition and arrangement of the components in the nucleosome.Problem 10
Describe the transitions that occur as nucleosomes are coiled and folded, ultimately forming a chromatid.Problem 11
Provide a comprehensive definition of heterochromatin and list as many examples as you can.Problem 12
Mammals contain a diploid genome consisting of at least 10⁹ bp. If this amount of DNA is present as chromatin fibers, where each group of 200 bp of DNA is combined with 9 histones into a nucleosome and each group of 6 nucleosomes is combined into a solenoid, achieving a final packing ratio of 50, determine (a) the total number of nucleosomes in all fibers, (b) the total number of histone molecules combined with DNA in the diploid genome, and (c) the combined length of all fibers.Problem 13
Assume that a viral DNA molecule is a 50-µm-long circular strand with a uniform 20-Å diameter. If this molecule is contained in a viral head that is a 0.08-µm-diameter sphere, will the DNA molecule fit into the viral head, assuming complete flexibility of the molecule? Justify your answer mathematically.Problem 14
How many base pairs are in a molecule of phage T2 DNA 52-µm long?Problem 15
Examples of histone modifications are acetylation (by histone acetyltransferase, or HAT), which is often linked to gene activation, and deacetylation (by histone deacetylases, or HDACs), which often leads to gene silencing typical of heterochromatin. Such heterochromatinization is initiated from a nucleation site and spreads bidirectionally until encountering boundaries that delimit the silenced areas. Recall from earlier in the text (see Chapter 4) the brief discussion of position effect, where repositioning of the w⁺ allele in Drosophila by translocation or inversion near heterochromatin produces intermittent w⁺ activity. In the heterozygous state (w⁺/w) a variegated eye is produced, with white and red patches. How might one explain position-effect variegation in terms of histone acetylation and/or deacetylation?Problem 20
In a study of Drosophila, two normally active genes, w⁺ (wild-type allele of the white-eye gene) and hsp26 (a heat-shock gene), were introduced (using a plasmid vector) into euchromatic and heterochromatic chromosomal regions, and the relative activity of each gene was assessed [Sun et al. (2002)]. An approximation of the resulting data is shown in the following table. Which characteristic or characteristics of heterochromatin are supported by the experimental data? Gene Activity (relative percentage) _ Euchromatin Heterochromatin hsp26 100% 31% w⁺ 100% 8%Problem 21
While much remains to be learned about the role of nucleosomes and chromatin structure and function, recent research indicates that in vivo chemical modification of histones is associated with changes in gene activity. One study determined that acetylation of H3 and H4 is associated with 21.1 percent and 13.8 percent increases in yeast gene activity, respectively, and that histones associated with yeast heterochromatin are hypomethylated relative to the genome average [Bernstein et al. (2000)]. Speculate on the significance of these findings in terms of nucleosome–DNA interactions and gene activity.Problem 22
An article entitled 'Nucleosome Positioning at the Replication Fork' states: 'both the 'old' randomly segregated nucleosomes as well as the 'new' assembled histone octamers rapidly position themselves (within seconds) on the newly replicated DNA strands' [Lucchini et al. (2002)]. Given this statement, how would one compare the distribution of nucleosomes and DNA in newly replicated chromatin? How could one experimentally test the distribution of nucleosomes on newly replicated chromosomes?Problem 24
Following is a diagram of the general structure of the bacteriophage chromosome. Speculate on the mechanism by which it forms a closed ring upon infection of the host cell.Problem 27
Spermatogenesis in mammals results in sperm that have a nucleus that is 40 times smaller than an average somatic cell. Thus, the sperm haploid genome must be packaged very tightly, yet in a way that is reversible after fertilization. This sperm-specific DNA compaction is due to a nucleosome-to-nucleoprotamine transition, where the histone-based nucleosomes are removed and replaced with arginine-rich protamine proteins that facilitate a tighter packaging of DNA. In 2013 Montellier et al. showed that replacement of the H2B protein in the nucleosomes with a testis-specific variant of H2B called TSH2B is a critical step prior to the nucleosome-to-nucleoprotamine transition. Mice lacking TSH2B retain H2B and their sperm arrest late in spermatogenesis with reduced DNA compaction. Based on these findings, would you expect that TSH2B-containing nucleosomes are more or less stable than H2B-containing nucleosomes? Explain your reasoning.Problem 30
Because of its rapid turnaround time, fluorescent in situ hybridization (FISH) is commonly used in hospitals and laboratories as an aneuploid screen of cells retrieved from amniocentesis and chorionic villus sampling (CVS). Chromosomes 13, 18, 21, X, and Y (see Chapter 8) are typically screened for aneuploidy in this way. Explain how FISH might be accomplished using amniotic or CVS samples and why the above chromosomes have been chosen for screening.Problem 31
To gauge the fidelity of DNA synthesis, Arthur Kornberg and colleagues devised a technique called nearest-neighbor analysis, which determines the frequency with which any two bases occur adjacent to each other along the polynucleotide chain (J. Biol. Chem. 236: 864–875). This test relies on the enzyme spleen phosphodiesterase (see the previous problem). As we saw in Figure 11-8, DNA is synthesized by polymerization of 5'-nucleotides—that is, each nucleotide is added with the phosphate on the of deoxyribose. However, as shown in the accompanying figure, the phosphodiesterase enzyme cleaves DNA between the phosphate and the C-5' atom, thereby producing 3'-nucleotides. In this test, the phosphates on only one of the four nucleotide precursors of DNA (cytidylic acid, for example) are made radioactive with ³²P and DNA is synthesized. Then the DNA is subjected to enzymatic cleavage, in which the radioactive phosphate is transferred to the base that is the 'nearest neighbor' on the 5' side of all cytidylic acid nucleotides. Following four separate experiments, in each of which a different one of the four nucleotide types is radioactive, the frequency of all 16 possible nearest neighbors can be calculated. When Kornberg applied the nearest-neighbor frequency test to the DNA template and resultant product from a variety of experiments, he found general agreement between the nearest-neighbor frequencies of the two. Analysis of nearest-neighbor data led Kornberg to conclude that the two strands of the double helix are in opposite polarity to one another. Demonstrate this approach by determining the outcome of such an analysis if the strands of DNA shown here are (a) antiparallel versus (b) parallel:
