7. DNA and Chromosome Structure

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.

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. In 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?

The accompanying chromosome diagram represents a eukaryotic chromosome prepared with Giemsa stain. Indicate the heterochromatic and euchromatic regions of the chromosome, and label the chromosome's centromeric and telomeric regions.

Are you more likely to find the DNA sequence encoding the digestive enzyme amylase in a heterochromatic, euchromatic, centromeric, or telomeric region? Explain your reasoning.

The accompanying chromosome diagram represents a eukaryotic chromosome prepared with Giemsa stain. Indicate the heterochromatic and euchromatic regions of the chromosome, and label the chromosome's centromeric and telomeric regions.

Why are expressed genes not found in the telomeric region of chromosomes?

The accompanying chromosome diagram represents a eukaryotic chromosome prepared with Giemsa stain. Indicate the heterochromatic and euchromatic regions of the chromosome, and label the chromosome's centromeric and telomeric regions.

Do you expect the centromeric region to contain heterochromatin? Why or why not?

The accompanying chromosome diagram represents a eukaryotic chromosome prepared with Giemsa stain. Indicate the heterochromatic and euchromatic regions of the chromosome, and label the chromosome's centromeric and telomeric regions.

What term best describes the shape of this chromosome?

Histone protein H4 isolated from pea plants and cow thymus glands contains 102 amino acids in both cases. A total of 100 of the amino acids are identical between the two species. Give an evolutionary explanation for this strong amino acid sequence identity based on what you know about the functions of histones and nucleosomes.

A survey of organisms living deep in the ocean reveals two new species whose DNA is isolated for analysis. DNA samples from both species are treated to remove nonhistone proteins. Each DNA sample is then treated with DNase I that cuts DNA not protected by histone proteins but is unable to cut DNA bound by histone proteins. Following DNase I treatment, DNA samples are subjected to gel electrophoresis, and the gels are stained to visualize all DNA bands in the gel. The staining patterns of DNA bands from each species are shown in the figure. The number of base pairs in small DNA fragments is shown at the left of the gel. Interpret the gel results in terms of chromatin organization and the spacing of nucleosomes in the chromatin of each species.

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.

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?

Experimental evidence demonstrates that the nucleosomes present in a cell after the completion of S phase are composed of some 'old' histone dimers and some newly synthesized histone dimers. Describe the general design for an experiment that uses a protein label such as ³⁵S to show that nucleosomes are often a mixture of old and new histone dimers following DNA replication.

DNase I cuts DNA that is not protected by bound proteins but is unable to cut DNA that is complexed with proteins. Human DNA is isolated, stripped of its nonhistone proteins, and mixed with DNase I. Samples are removed after 30 minutes, 1 hour, and 4 hours and run separately in gel electrophoresis. The resulting gel is stained to make all DNA fragments in it visible, and the results are shown in the figure. DNA fragment sizes in base pairs (bp) are estimated by the scale to the left of the gel. Draw a conclusion about the organization of chromatin in the human genome from this gel.

DNase I cuts DNA that is not protected by bound proteins but is unable to cut DNA that is complexed with proteins. Human DNA is isolated, stripped of its nonhistone proteins, and mixed with DNase I. Samples are removed after 30 minutes, 1 hour, and 4 hours and run separately in gel electrophoresis. The resulting gel is stained to make all DNA fragments in it visible, and the results are shown in the figure. DNA fragment sizes in base pairs (bp) are estimated by the scale to the left of the gel. Examine the gel results and speculate why longer DNase I treatment produces different results.

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.