Deck 11: Analyzing Genomic Variation

Question 1

For each of the terms in the left column, choose the best matching phrase in the right column.

Answer 1

a.Telomeres are specialized repeating sequences at the end of every linear chromosome in eukaryotes, which does not code for any protein. In humans, the telomeric base sequence found is 5' TTAGGG 3'.Thus, the option (a), matches with the option 4.b.
G bands (Giemsa bands) are formed by staining the chromosomes with Giemsa stain. Alternate light and dark bands are formed of which the lighter bands are known as G bands.Thus, the option (b) matches with the option 10.c.Kinetochore is the point of attachment of the chromosome to the spindle fibers. This region contains of a complex of proteins and DNA (deoxyribonucleic acid).Thus, the option (c) matches with the option 7.d.A nucleosome is a bead like structure primarily made up of DNA, which is wound around histone proteins. Each of the nucleosome consists of one half of histone dimer and one half of histone tetramers.Thus, the option (d) matches with the option 8.e.ARS (autonomously replicating sequence) is the yeast site of origin of replication and is one of the three important parts that is needed to make an artificial chromosome.Thus, the option (e) matches with the option 2.
f.Satellite DNA are found only in higher eukaryotes. They are areas of repetitive DNA found near the centromeres.Thus, the option (f) matches with the option 3.g.Chromatin is thread like structures found in the nucleus of eukaryotes made up of DNA, protein and RNA (ribonucleic acid).Thus, the option (g) matches with the option 5.h.Cohesin is a protein complex that binds with the sister chromatids from prophase and keeps them together until anaphase when it is broken down by separase enzyme.Thus, the option (h) matches with the option 1.i.Histones are proteins that form the basic structure of a nucleosome. They form the core of the nucleosome by binding with the DNA.Thus, the option (i) matches with the option 6.j.Shelterin is a protein that coats with the telomeres and forms a complex with them. This complex protects the telomeres from end fusion and degradation by various enzymes.Thus, the option (j) matches with the option 9.

Question 2

Many proteins other than histones are found associated with chromosomes. What roles do these nonhistone proteins play Why are there more different types of nonhistone than histone proteins

Answer 2

Non-histone proteins are a heterogeneous group of proteins that make up half the mass of proteins associated with chromosome. There are hundreds and thousands of different kinds of nonhistone chromosomal proteins.
These large variety of nonhistone proteins perform a variety of functions. Some of these include:
• They form structural backbone helping to package deoxyribonucleic acid (DNA) into more complex structures. E.g. Scaffold proteins.
• Some nonhistone proteins are active in replication of DNA. E.g. DNA polymerase.• They play an important role in chromosomal segregation. E.g. motor proteins of kinetochores help the movement of chromosome along the spindle apparatus and helps in their movement from parent to daughter cells during mitosis and meiosis.
• They also help to regulate transcription and ribonucleic acid (RNA) processing at the time of gene expression.
Unlike histone proteins, nonhistone proteins play important roles in higher order chromosome organization, differential chromosome packaging, chromatin architecture, replication, segregation, etc. Since they perform a wide variety of functions compared to histone proteins, they are more in number.

Question 3

What difference is there in the compaction of chromosomes during metaphase and interphase Give at least one reason why this difference may be necessary.

Answer 3

Chromosome condensation is an event in both mitosis and meiosis. It is the shortening of chromosomes axis and contraction of radius. It results in diminished chromosome volume. In interphase, the chromosomes undergo a 40-fold compaction, while in metaphase they undergo a 10,000-fold compaction. Therefore, chromosomes are less compact in interphase than metaphase in eukaryotes.
The difference in compaction is necessary, because at only in 10,000-fold compaction that the centromeres and kinetochores become clear that are necessary for the chromatids to attach themselves to the spindle fibers. Therefore, the segregation of chromosomes would take place properly into opposite poles.

Question 4

What is the role of the core histones in compaction compared to the role of histone H1

Question 5

a. About how many molecules of histone H2A would be required in a typical human cell just after the completion of S phase, assuming an average nucleosome spacing of 200 bp
b. During what stage of the cell cycle is it most crucial to synthesize new histone proteins
c. The human genome contains 60 histone genes, with 10-15 genes of each type (H1, H2A, H2B, H3, and H4). Why do you think the genome contains multiple copies of each histone gene

Question 6

The enzyme micrococcal nuclease can cleave phosphodiester bonds on single- or double-stranded DNAs, but DNA that is bound to proteins is protected from digestion by micrococcal nuclease. When chromatin from eukaryotic cells is treated for a short period of time with micrococcal nuclease and then the DNA is extracted and analyzed by electrophoresis and ethidium bromide staining, the pattern shown in lane A on the gel below is found. Treatment for a longer time results in the pattern shown in lane B, and treatment for yet more time yields that shown in lane C. Interpret these results.

Question 7

a. What letters are used to represent the short and long arms of human chromosomes
b. Sketch a schematic diagram of a hypothetical chromosome 3 that has 3 regions with 2 bands each on the short arm and 5 regions with 3 bands each on the long arm. Label the arms, regions, and bands and indicate a gene at position 3p32.

Question 8

About 2000 G bands are visible in a high-resolution karyotype of the 3 billion base pairs in the haploid human genome. If the genome contains about 25,000 genes, about how many genes would be removed by a deletion of DNA that could be detected by karyotype analysis

Question 9

Suppose you performed a fluorescence in situ hybridization experiment (FISH) on chromosomes from a human cell using a probe corresponding to a gene located near (but not at) the telomere of the q arm of chromosome 4.
a. On the following idiogram, which shows only chromosomes 1-5 contained in this diploid cell, indicate the location of all fluorescent signals.
b. Compare your idiogram with the result of the FISH experiment shown in Fig. 11.9a. Why are the chromosomes scattered in Fig. 11.9a, rather than being present in neatly arranged pairs of homologous chromosomes as in the idiogram Do you think it is likely that the gene whose DNA was used as a probe in Fig. 11.9a is found on the q arm of human chromosome 4
Figure 11.9 Fluorescent In situ hybridization. (a) FISH analysis of a human cell's chromosomes. The four yellow spots show where a probe made from a angle gene hybridizes to the two sister chromatids on each of two homologous chromosomes. (b) Spectral karyotyping (SKY). Probesmade from DNA along the length of each chromosome are labeled with fluorescent tags of different colors.
(a)
(b)

Question 10

Which of the following would be suggested by a DNase hypersensitive site
a. No transcription occurs in this region of the chromosome.
b. The chromatin is in a more open state than a region without the hypersensitive site.
c. Transcription terminates at this site.

Question 11

For each of the following pairs of chromatin types, which is the most condensed
a. 100 Å fiber or 300 Å fiber
b. 300 Å fiber or DNA loops attached to a scaffold
c. Euchrmatin or heterochromatin
d. Interphase chromosomes or metaphase chromosomes

Question 12

Give examples of constitutive and facultative heterochromatin in
a. Drosophila
b. humans

Question 13

One histone modification that is seen consistently in many species is the addition of an acetyl group to the twelfth lysine in the H4 protein. If you were a geneticist working on yeast and had a clone of the H4 gene, what could you do to test whether the acetylation at this specific lysine was necessary for the functioning of chromatin

Question 14

Drosophila geneticists have isolated many mutations that modify position-effect variegation. Dominant suppressors of variegation [ Su(var)s ] cause less frequent inactivation of genes brought near heterochromatin by chromosome rearrangements, while dominant enhancers of variegation [ E(var )s] cause more frequent inactivation of such genes.
a. What effects would each of these two kinds of mutations have on position-effect variegation of the white gene in Drosophila.
b. Assuming that these Su(var) and E(var) mutations are loss-of-function (null) alleles in the corresponding genes, what kinds of proteins do you think these genes encode

Question 15

On the following figures, genes A and B are on the X chromosome ( blue ) and both are subject to X inactivation, while genes C and D are on chromosome 17 (an autosome; red ). F and S refer to alleles encoding fast-and slow-migrating forms of the corresponding proteins that can be discriminated by electrophoresis. For women 2 and 3 in the figures that follow, indicate all the possible forms of the four proteins that could be expressed in clones made from different individual somatic cells that already had one or more Barr bodies. As an example, some clones from normal woman 1 could express the A F , B F , C F , C S , D F , and D S proteins, while other clones could express the A S , B S , C F , C s , D F and D S proteins. None of the clones from woman 1 should make both the slow and fast forms of proteins A or B. Woman 2 is a heterozygote for a deletion of the XIC. Woman 3 is a heterozygote for a reciprocal translocation in which parts of the X chromosome and chromosome 17 have exchanged places.

Question 16

How one X is chosen to express Xist is unknown. One clue to the choice mechanism lies in another gene in the XIC called Tsix, which is transcribed only from the X chromosome that will remain active. Tsix overlaps Xist and is transcribed in the opposite direction, as shown in the figure below. Tsix produces a long noncoding RNA whose sequence is complementary (antisense) to that of Xist , explaining its name (" Xist " backwards is " Tsix "). Tsix expression from the future active X prevents Xist expression from that X chromosome.
a. Suppose a female mammal has one normal X chromosome and one X chromosome that has a mutation preventing expression of the Tsix RNA but allowing expression of the Xist RNA. In the cells of this female, which X chromosome would be more likely to become a Barr body
b. Formulate a hypothesis that might explain why Tsix transcription on an X chromosome might interfere with the expression of Xist from the same X chromosome.
c. Why does this clue about Tsix still not solve the problem about how cells decide which X chromosomes to inactivate by heterochromatization

Question 17

The human genome contains about 3 billion base pairs. During the first cell division after fertilization of a human embryo, S phase is approximately 3 hours long. Assuming an average DNA polymerase rate of 50 nucleotides/sec over the entire S phase, what is the minimum number of origins of replication you would expect to find in the human genome

Question 18

The mitotic cell divisions in the early embryo of D. melanogaster occur very rapidly (every 8 minutes).
a. If there was one bidirectional origin in the middle of each chromosome, how many nucleotides would DNA polymerase have to add per second to replicate all the DNA in the longest chromosome (66 Mb) during the 8-minute early embryonic cell cycles (Assume that replication occurs during the entire cell division cycle.)
b. In fact, many origins of replication are active on each chromosome during the early embryonic divisions and are spaced approximately 7 kb apart. Calculate the average rate (per second) with which DNA polymerase adds complementary nucleotides to a growing chain in the early Drosophila embryo.

Question 19

a. What DNA sequences are found at the telomeres of human chromosomes
b. What functions do the two telomere-associated complexes, telomerase and shelterin, fulfill at chromosome ends

Question 20

a. Mice engineered to block expression of the gene encoding the telomerase protein age at a much faster rate than normal and have decreased life spans. When expression of the telomerase protein is turned back on in mice that are prematurely old, many negative effects of this aging are rapidly and dramatically reversed. Provide a possible explanation for these results.
b. The results of these mouse experiments have led some researchers to propose that treatments that could lead to overexpression of the telomerase gene might serve as a "fountain of youth" leading to reversal of aging in humans. Why do you think we should be very cautious about trying such treatments Your argument should address why it might be advantageous to multicellular organisms for most of their somatic cells not to express telomerase.

Question 21

a. In a fluorescent in situ hybridization (FISH) experiment, what would you see if you used DNA containing multiple copies of 3 ' AATCCC 5 ' as a probe Show your results on the idiogram accompanying Problem 9 on p. 404.
b. What DNA sequence would you use as a probe to track one end of one particular chromosome in a FISH experiment What would your results look like on the same idiogram
Problem 9
Suppose you performed a fluorescence in situ hybridization experiment (FISH) on chromosomes from a human cell using a probe corresponding to a gene located near (but not at) the telomere of the q arm of chromosome 4.
a. On the following idiogram, which shows only chromosomes 1-5 contained in this diploid cell, indicate the location of all fluorescent signals.
b. Compare your idiogram with the result of the FISH experiment shown in Fig. 11.9a. Why are the chromosomes scattered in Fig. 11.9a, rather than being present in neatly arranged pairs of homologous chromosomes as in the idiogram Do you think it is likely that the gene whose DNA was used as a probe in Fig. 11.9a is found on the q arm of human chromosome 4
Figure 11.9 Fluorescent In situ hybridization. (a) FISH analysis of a human cell's chromosomes. The four yellow spots show where a probe made from a angle gene hybridizes to the two sister chromatids on each of two homologous chromosomes. (b) Spectral karyotyping (SKY). Probesmade from DNA along the length of each chromosome are labeled with fluorescent tags of different colors.
(a)
(b)

Question 22

If you are comparing the two telomeres in each entry in the following list, in which cases would you expect the two telomeres always to have exactly the same number of TTAGGG repeats
a. One telomere at one end of a chromosome, one telomere at one end of a nonhomologous chromosome.
b. One telomere at one end of a chromosome, one telomere at the corresponding end of the homologous chromosome.
c. One telomere at one end of a chromosome, the other telomere at the other end of the same chromosome.
d. One telomere at one end of a chromatid, the other telomere at the corresponding position in the sister chromatid.

Question 23

a. What DNA sequences are commonly found at human centromeric regions
b. What functions do the two centromere-associated complexes, cohesin and the kinetochore, play in chromosome mechanics

Question 24

The Rec8 protein is a cohesin complex subunit that is normally made only during meiosis; it substitutes for the purple protein shown in the mitotic cohesin complex in Fig. 11.25 on p. 400. Rec8 is not cleaved during meiosis I but is cleaved during meiosis II to finally allow sister chromatids to segregate.
Scientists hypothesized that a protein (shugoshin) protects the Rec8 protein from cleavage and degradation during meiosis I. To identify shugoshin, researchers first produced the Rec8 protein in mitotically dividing yeast cells. In these cells, Rec8 was cleaved during mitosis and the cells suffered no harmful effects. Researchers then expressed other, normally meiosis-specific proteins in cells expressing Rec8 mitotically. The scientists were able to identify shugoshin as a protein that protects Rec8 from degradation.
What effect do you think expressing shugoshin had on the mitotically dividing cells expressing Rec8 What phenotype would the cells show
Figure 11.25 A molecular model for cohesin. The cohesion complex has protein subunits ( green , aqua , and purple ) that together surround the two sister chromatids (the two DNA molecules with strands In blue and red ). Sister chromatids can separate when a protease (separase; gold ) cleaves the purple cohesin subunit at the site shown by the arrow , freeing the two DNA molecules.

Question 25

In the following diagram, each line represents a double-stranded DNA molecule.
a. What type of cell division is being represented, and which stages of that cell division are shown in the two parts of the figure What is the relationship between the lines drawn in the same shade (light or dark) of blue Between lines drawn in different shades of blue
b. Cohesin is added to chromosomes immediately after S phase. Cohesin complexes are particularly concentrated at the centromeres, but there are also some scattered cohesin complexes located along the chromosome arms. On a copy of the figure, indicate the distribution of cohesin complexes on the chromosomes. Distinguish between cohesin at the centromeres and cohesin along the arms. Your diagram should represent how cohesin might keep DNA molecules together.
c. Look carefully at your drawings. What keeps all the blue lines together at the metaphase plate of this kind of cell division, even though forces are pulling at the kinetochores in opposite directions
d. Again looking carefully at your drawings, what can you conclude about the function of shugoshin during the type of cell division being depicted What is the name of the enzyme whose function shugoshin prevents, and what does this enzyme do

Question 26

In the 1920s, Barbara McClintock, later a Nobel laureate for her discovery of transposable elements, examined the behavior of chromosomes in wheat cells that had been subjected to X-rays. She noticed that the X-rays produced chromosomal breaks during G1 phase, and that after subsequent chromosome replication in S phase, the broken ends of the two sister chromatids could join together to make a "fusion" chromosome larger than the original. Even later, during mitotic metaphase and early in mitotic anaphase, the joined sister chromatids would form an unusual "bridge" structure in which chromatin was stretched between the two spindle poles and could then eventually break. She called this phenomenon the breakage-fusion-bridge cycle. Each of photographs (a) and (b) that follow shows a cell in early mitotic anaphase that has two such chromatin bridges.
a. What ensures that the ends of normal chromosomes do not fuse together as do the ends of the sister chromatids after breakage
b. The following figure shows a chromosome with genes A - G ; the arrow indicates the location of X-ray-induced breakage. Draw the resulting bridge (that is, the large fused chromosome) as it would be seen in mitotic anaphase, and label all the genes and important chromosomal structures the bridge contains. Use arrows to show the forces exerted by the spindle apparatus on this bridge.
c. If the sister chromatids fuse, why must the fusion chromosome behave as a bridge during mitosis (Think about the forces pulling on the bridge described in your answer to part [b].)
d. What is likely to happen to the bridge during mitotic anaphase What then is likely to happen in the two daughter cells produced by the mitosis just described, and why (Hint: McClintock's name for this phenomenon implies it is a cycle.)

Question 27

Give at least one example of a chromosomal structure or function affected by the following mechanisms for regulating chromatin structure.
a. Posttranslational changes of the normal histones found in the nucleosome
b. Nucleosomes with variant histones that are encoded by special genes

Question 28

a. Give at least three examples of types of mutations that would disrupt the process of mitotic chromosome segregation. That is, explain in what DNA structures or in genes encoding what kinds of proteins would you find these segregation-disrupting mutations.
b. How could you use yeast artificial chromosomes (YACs) to find such mutations in S. cerevisiae

Question 29

A number of yeast-derived elements were added to the circular bacterial plasmid pBR322. Yeast that require uracil for growth (Ura cells) were transformed with these modified plasmids and Ura + colonies were selected by growth in media lacking uracil. For plasmids containing each of the elements listed in parts (a-c), indicate whether you expect the plasmid to integrate into a chromosome by recombination, or instead whether it is maintained separately as a plasmid. If the plasmid is maintained autonomously, is it stably inherited by all of the daughter cells of subsequent generations when you no longer select for Ura + cells (that is, when you grow the yeast in media containing uracil)
a. URA + gene
b. URA + gene, ARS
c. URA + gene, ARS, CEN (centromere)
d. What would need to be added in order for these sequences to be maintained stably in yeast cells as a linear artificial chromosome

Question 30

A DNA fragment containing yeast centromere DNA was cloned into a TRP ARS plasmid, YRp7, causing the plasmid to become mitotically very stable (that is, the plasmid was transmitted during mitotic divisions to each daughter cell). The assay for mitotic stability consists of growing a transformed cell without selection for the plasmid and determining the number of Trp + colonies remaining after 20 generations of growth under conditions that are not selective for the plasmid. To identify the region of the cloned fragment that contained centromere DNA, you cut the initial fragment into smaller pieces, reclone those pieces into YRp7, and test for mitotic stability. Based on the map that follows and results of the mitotic stability assay, where is the centromeric DNA located

Question 31

Diagram the DNA components of a chromosome, including the polarity of strands.

Question 32

Contrast histone and nonhistone proteins in terms of structure and function.

Question 33

Diagram the structure of a nucleosome.

Question 34

Describe nucleosome supercoiling and its relationship to the radial loop-scaffold model of chromatin packaging.

Question 35

Summarize the process of producing G bands in a chromosome and how these bands are used in locating genes.

Question 36

Describe FISH analysis and its application in finding specific DNA sequences in a chromosome.

Question 37

Describe how chromatin remodeling complexes allow gene expression to occur.

Question 38

Differentiate between gene expression in heterochromatic and euchromatic regions.

Question 39

Describe how scientists use position effect variegation to study the mechanisms underlying formation of heterochromatin.

Question 40

Outline how histone methylation and acetylation affect chromatin structure and gene expression.

Question 41

Summarize the role of the Xist gene in X inactivation in mammalian cells.

Question 42

Explain how replication of the DNA in long eukaryotic chromosomes can occur in a short period of time.

Question 43

Summarize the process by which nucleosomes are formed during replication.

Question 44

Discuss the structure of telomeres and their role in maintaining chromosome integrity.

Question 45

Describe the action of telomerase and identify the cell types in which it continues to be synthesized.

Question 46

Contrast the DNA sequences dictating centromere formation in yeast and in higher eukaryotes.

Question 47

Describe spindle attachment during metaphase and the role tension on kinetochores plays in ensuring proper chromosome segregation.

Question 48

Compare the behavior of cohesin complexes during mitosis, meiosis I, and meiosis II.

Question 49

List the elements that must be included in an artificial chromosome.

Question 50

Describe the consequences of constructing recombinant DNA molecules that lack any of these elements.