Organization and Packaging of Chromosomal DNA

Question 1

What phase of the cell cycle is the cell mainly in?

Answer 1

Interphase

Question 2

In interphase, what form are the chromosomes in?

Answer 2

The chromatin is in a very extended form, whereas the chromosomes are most highly condensed in mitosis

Question 3

In what phase does DNA replication occur?

Answer 3

Interphase

Question 4

What are the three types of DNA sequences that must be present in order for replication to occur?

Answer 4

1) Origin of Replication
2) Centromere
3) Telomeres

Question 5

Define the centromere

Answer 5

It is the most highly condensed region of the chromosome and allows the kinetochores to bind to it to pull apart chromosomes during mitosis.

Question 6

Interphase

Answer 6

Have 2 copies of each chromosome except for sex chromosomes

Chromosomes here do not look like the “typical” chromosome. They are not as highly condensed so that DNA replication and gene expression can occur

This is where you have gene expression and DNA replication

The copies that were replicated (sister chromatids) are near each other, then they go through mitosis to be separated.

Question 7

Mitosis

Answer 7

The centromeres get attached to the spindles and get pulled apart in cell division.

If you were to look at a chromosome in this stage, it would look like the “typical” chromosome with 2 sister chromatids joined at the centromere. These were generated from replication in interphase. It is the most highly compacted from of DNA.

Question 8

Chromosome Visualization

Answer 8

Multiple techniques have been developed to visualize chromosomes and determine chromosome abnormalities associated with particular diseases. It has also shown recently that chromosomes localize to subnuclear domains, even during interphase.

Question 9

Chromosome painting or Spectal karyotyping

Answer 9

Stains each chromosome a different color to distinguish them from one another. It is done by making probes that are complementary to the DNA and essentially span the entire chromosome. This can be done in interphase as well. This is how they showed that interphase chromosomes were actually very organized

Question 10

Giemsa-staining

Answer 10

When done in early mitotic chromosomes it gives a characteristic banding pattern. It gives a unique barcode to distinguish one region of the chromosome from another.

Question 11

Karyotype

Answer 11

Arrangement of the full chromosome set it numerical order

Question 12

Chromatin

Answer 12

It is DNA plus the associated proteins

It is made up of 1/3 DNA by mass and 2/3 proteins

1/3 of the protein mass are histone proteins

Question 13

Genes

Answer 13

DNA sequences that produce a functional RNA molecule (encode for a protein or forms a structural or regulatory RNA).

comprised of introns, exons, and regulatory regions.

A very small amount of the gene will actually code for protein. Much of it is introns and regulatory DNA.

Question 14

Exons

Answer 14

Coding regions of the DNA. ~1.5% of DNA sequence is in exons.

Question 15

Introns

Answer 15

Unique sequences that don’t code for proteins or don’t contain genes.

Question 16

Distinguish different types of DNA sequences

Answer 16

1/2 of the genome is unique sequences (introns, exons, etc.) and 1/2 of the genome is repetitive DNA (transposable elements, regulatory sequences, etc.)

Question 17

Describe the components and structural organization of the Nucleosome

Answer 17

It is the histone proteins and the DNA wrapped around it. Doesn’t include the linker DNA. The Nucleosome is comprised of an octameric histone core composed of 2 of each of the following: H2A, H2B, H3, and H4. There is roughly 147 nucleotides that surround the histone core and wrap around it 1.67 times. This DNA is more resistant to nuclease activity because the proteins protect it. Serve multiple functions!

Question 18

Linker DNA

Answer 18

DNA not wrapped around proteins and thus it is more susceptible to digestion

Question 19

Naked DNA

Answer 19

DNA not associated with proteins. Diameter is 2nm

Question 20

“Beads-on-a-string”

Answer 20

It is chromatin that is not wrapped. Diameter is 11nm

Question 21

30nm Fiber

Answer 21

The nucleosomes begin to wrap around one another. This is how interphase chromosomes are structured. Diameter is 30nm. Can form a zig-zag or solenoid structure. This compaction is mediated by histone tail interactions and H1 proteins.

Question 22

“Looping” Structure: Sections of Chromosomes in extended form

Answer 22

300nm diameter. The 30nm fibres begin to “loop”

Question 23

Condensed section of chromosome

Answer 23

700nm diameter

Question 24

Mitotic Chromosome

Answer 24

1400nm diameter. Net result is it is 10,000-fold shorter.

Question 25

Compare features of highly condensed and less condensed chromatin

Answer 25

More condensed chromatin is shorter but has a greater width. Also, this is likely to be heterochromatin which is less likely to be actively expressed. Less condensed chromatin is longer and has a smaller width. These is likely to be euchromatin and more expressed or more active.

Question 26

Explain Mechanisms for dynamic change in chromosomes

Answer 26

Nucleosomes are dynamic and are often changed by ATP-dependent chromatin remodelling complexes which can slide or remove nucleosomes, or exchange histone proteins. One group of chromatin remodelling complexes slide or move histones while another degrades then or exchanges them. Both require ATP

Question 27

Histone Fold

Answer 27

3 alpha helices that conform into a structure like a hand that conforms with another triple helix molecule to form a hand-shake like structure. This is how H2A and H2B come together to form a dimer and H3 and H4 come together to form a dimer and later a tetramer.

Question 28

Histone Tail

Answer 28

Each core histone protein also has an amino-terminal tail that is very important for gene regulation. They are subject to covalent modifications that can affect chromatin structure and function. The histone amino-terminal tails can also interact with the amino-terminal tail of another histone in another histone (from another nucleosome) to help compact the chromatin.

Question 29

Understand the roles of histone H1, inter-nucleosome tail interactions and condensins in chromatin compaction

Answer 29

These mediate chromatin compaction.

Histone H1 binds to where the DNA enters and exits the histone core protein. It brings the parts of the DNA just enough together to help compact the DNA even more.

Inter-nuclesome tail: The tails from adjacent nucleosomes interact to help bring the nucleosomes more together, again resulting in further compaction.

Question 30

Where would one likely find highly active DNA within the nucleus?

Answer 30

towards the interior of the nucleus

Question 31

Where would one likely find genes silenced in heterochromatin in the nucleus?

Answer 31

Towards the periphery of the nucleus

Question 32

Fractal Globule Model

Answer 32

It is a knot-free conformation that shows dense packing of DNA and retains the ability to fold and unfold for gene expression. Regions that need to be expressed are not all knotted up, they can be easily accessible. Each chunk of DNA is organized into its own domain.

Question 33

Heterochromatin

Answer 33

Highly folded DNA. It either contains no genes (centromeres and telomeres) or is resistant to gene expression. Heterochromatin is dynamic and tends to be self-sustaining once formed. If genes are relocated to heterochromatin, they are silenced. During development, heterochromatin can spread if the special boundary sequences aren’t present.

Question 34

Genetic Inheritance

Answer 34

It is a permanent mutation. It is passed down to generations. It passes down into germ cells.

Question 35

Epigenetic Inheritance

Answer 35

It is not passed down to offspring. It is a change in the chromatin structure that results in turning a gene off. You are not changing the DNA sequence here, rather it is changing how the DNA is packaged. In somatic cells this information tends to stay the same but in germ cells the information is erased so it tends to go away.

Question 36

3 Models for Barrier Proteins blocking the spread of heterochromatin to adjacent euchromatin regions

Answer 36

1) Physical barrier: A protein binds to a region of the DNA and then another portion of that protein will bind to another structure, like the nuclear pore, and prevent the spreading of one type of epigenetic information across the barrier
2) Coats Nucleosomes: Another barrier covers or coats the nucleosomes so the heterochromatin can’t spread.
3) Barrier with Enzymatic activity: Acts on histones of heterochromatin to prevent them from modifying the adjacent histones.

These are different types of barriers but they are all functioning in the same fashion.

Question 37

Recognize common covalent modifications of histone proteins

Answer 37

Amino-terminal tails are available to the outside environment so they can be acted on or act on something else. N-terminal tails ALL have Lysine which is basic and thus positively charged. This allows it to react with the negatively charged phosphodiester backbone of DNA. These Lysines can also be post-translationally modified to be acetylated or methylated but they cannot be both at the same time. Can only get non-modified lysine, acetylated lysine or methylated lysine. There are also serines in the tail that can be phosphorylated and Arginines that can be methylated. There are multiple different post-translation modifications that can occur on the tail and they all provide different information.

Question 38

Lysine Acetylation

Answer 38

It adds an acetyl group (-CO-CH3) to the nitrogen. In doing so, the NH3+ is now neutralized. Because of the loss of the + charge, lysine can no longer interact with the - charged phosphates in the phosphodiester backbone as tightly. Acetylation is found to only occur on lysine although the other modifications are not lysine specific.

Question 39

Lysine Methylation

Answer 39

Lysine can be methylated (adding CH3) up to three times, displacing the Hydrogens on the nitrogen with the methyl groups. The charge on lysine in this case remains positive but each of these modifications can provide different information for the histone code.

Question 40

Histone Code

Answer 40

Covalent modification and combinations of modifications to histone tails attract specific proteins to that stretch of chromatin, which execute appropriate functions. There are proteins that recognize these various post-translational modifications in the context of a nucleosome core protein and they do “something”

Question 41

Understand the concept of histone readers and writers, and the histone code

Answer 41

The histone code refers to the post-translational modifications that occur, such as methylation of the amino-terminal tail, which then recruits certain regulatory proteins.

When there are tail modifications, certain proteins bind to these modifications which are a part of the “reader complex”. This reader then recruits other proteins to carry out some function, possibly one with catalytic activity.

Readers and writers can also be involved in heterochromatin formation by further condensing the chromatin. For example, histone writers are recruited and modify a histone which then recruits a reader protein. The reader then recruits another writer than modifies the adjacent histone. This then gives another signal to recruit another reader protein. These readers then spread a wave of chromatin condensation.

Question 42

Recognize the role of histone variants

Answer 42

Histone variants also affect chromosome structure. There are variants of histones that contribute to different functions of the regions of DNA wrapped in those nucleosomes. Histone H3 is the most abundant Histone H3, however, there are variants of histone H3 that package into nucleosomes just like H3 but provide different information for the histone code. An example is the CENP-A variant of histone H3 that is only found in centromeres. It helps to make the centromeres more compact. However, if variants occur where they shouldn’t, it can result in mutations.

Question 43

Recognize unique features of centromeric heterochromatin

Answer 43

Centromeric heterochromatin contains the H3 variant CENP-A which causes the centromeres to be much more highly condensed as well as able to bind to the kinetochores.

Question 44

Explore the potential effects of changes in chromosome structure on cancer outcomes

Answer 44

By making heterochromatin less condensed, the drug do do many things. First, it could expose tumor suppressor genes which would help fight the cancer if they are turned on. Also, it could cause the DNA to be so messed up that it makes the cell apoptotic.