Lecture 1: Genome Constitution, Chromatin Structure and Organization

Question 1

When comparing the genome constitution, what does the c-value paradox state?

Answer 1

The c-value paradox states that a larger genome size does not not mean the organism will be complex. The genome size is independent from the complexity of the organism. However, observed research has shown that typically eukaryotes, which are the more complex organisms, tend to have larger genomes.

Question 2

What eukaryote cell organelles have circular DNA, and what did they evolve from?

Answer 2

Mitochondria and Chloroplasts. Evolved from prokaryotes

Question 3

What is the disadvantage of having a open molecule in eukaryotic DNA?

Answer 3

An open molecule is vulnerable to degradation at the ends because exonuclease enzymes can attack the DNA at any time.

Question 4

What are endonuclease enzymes?

Answer 4

Endonuclease enzymes are able to attack the DNA at any location, however, in order for this occur, specific conditions need to be met. Therefore, this type of degradation is rare.

Question 5

What challenges occur with DNA replication for bacterial DNA?

Answer 5

Bacterial DNA is a closed molecule (circular) and therefore the two daughter molecules remain interlocked after replication occurs and therefore a third party is required to separate the molecules (topoisomerase).

Question 6

What is the role of topoisomerase in Bacterial DNA replication?

Answer 6

Topoisomerase are nuclear enzymes that help to separate the two daughter molecules that are interlocked following replication.

Question 7

What are the two ways that prokaryote maintain chromosomes? What is the chromosome number in each way?

Answer 7

1. Nucleoid = Haploidy which means there is one molecule that contains all the genetic information
2. Plasmid = Multiple copies per cell

Question 8

What are the ways that eukaryotes maintain the chromosome number?

Answer 8

1. Haploidy (n) - single celled (ex. yeast, gametes)
2. Diploidy (2n) - two complete sets of chromosomes
3. Polyploidy (3n,4n, etc) - More than two complete sets of chromosomes (ex. Megakaryocytes)
4. Endomitosis - division of chromosomes that is not followed by division of the nucleus and that results in an increased number of chromosomes in the cell. Results in endopolyploidy (ex. human liver, placenta, drosophila salivary glands)

Question 9

What is the meaning of gene density?

Answer 9

Gene density defines the quantity of genes that are present in one region of the genome. This is important to know because the entire genome does not code of genes, but rather only specific regions code for genes. For humans, only 1% of the entire genome is the coding region (exons).

Question 10

What is the exonic and geneic region?

Answer 10

The exonic region consists of only exons and this region equates for about 1% of the entire human genome. The genic region is the entire region that codes for the gene including the promoters, exons, etc, this is about 3% of the human genome. The rest of the genome is non-coding (including the genic region that is not exons).

Question 11

Why is 1 gene able to code for multiple proteins?

Answer 11

This is because of alternative splicing. 60% of the human genome is alternatively spliced and 80% of the splices from this 60% change their protein sequence. Hence why there are ~ 50,000 proteins but only 20,000 genes.

Question 12

What are the 3 major structural elements for the chromosome?

Answer 12

1. Origin of replication
2. Centromere
3. Telomere

Question 13

What is the origin of replication in the chromosome?

Answer 13

The origin of replication is the site of initiation for replication. In eukaryotes, one chromosome can have multiple origins of replication. Each origin is found 30-40kb apart, and is generally in the noncoding region. For prokaryotes, there is only one site for replication initiation.

Question 13

What is the origin of replication in the chromosome?

Answer 13

The origin of replication is the site of initiation for replication. In eukaryotes, one chromosome can have multiple origins of replication. Each origin is found 30-40kb apart, and is generally in the noncoding region. For prokaryotes, there is only one site for replication initiation.

Question 14

What is the role of centromeres in the chromosome?

Answer 14

Centromeres are required for the correct segregation of the chromosomes following the replication of the DNA.
- They help to direct the formation of the protein complex kinetochore which binds to the spindle fibres (Microtubles) which then pull away the sister chromosomes.

Question 15

What is the role of telomeres in chromosome structure?

Answer 15

The telomeres are located at the two ends of the chromosome. A telomere protein is assembled at the telomere location which is used to form structure that is resistant to frequent recombination and DNA degradation at the ends of the chromosome.
- They also act as specialized origins of replication that help to replicate the ends of the chromosomes by recruiting the DNA polymerase known as telomerase
- They contain TG rich repeat regions which is what helps with the prevention of DNA degradation at the ends.

Question 16

What is recombination?

Answer 16

a process by which pieces of DNA are broken and recombined to produce new combinations of alleles

Question 17

What is the difference between mitosis and meiosis?

Answer 17

-Mitosis: Sister chromatids are pulled apart and there is equal division because we want to maintain the number of chromosomes (2n -> 2n and 2n)

• Meiosis: Chromosomes are pulled apart and there is reduction division. (2n -> n)

Question 18

Do chromosomes occupy specific territories in the nucleus?

Answer 18

Yes, they do occupy specific territories. Often, the active genes are found near the borders of the territories. Sometimes actives genes from many territories can cluster together in a interchromsomal territory which enriches transcription.

Question 19

Do chromosomes occupy specific territories in the nucleus?

Answer 19

Yes, they do occupy specific territories. Often, the active genes are found near the borders of the territories. Sometimes actives genes from many territories can cluster together in a interchromsomal territory which enriches transcription.

Question 20

What is a chromatin? How does it differ from chromosomes?

Answer 20

The chromatin is a mixture of DNA and proteins which are used to from chromosomes in the nucleus of a cell. It differs from chromosomes because the fibres of the chromatin are long, thin and uncoiled. They still need to undergo the condensation process which helps them transform to the condensed and compact chromosomes.

Question 21

Why is the DNA packaged into chromosomes?

Answer 21

The DNA is packaged into chromosomes because
- since the chromosome is condensed and compact, the DNA can easily fit in the cell
- It helps to protect the DNA from damage that can be done by enzymes (ie. nucleases)
- It allows for efficient transfer to the daughter cells during cell division

Question 22

What is the packing ratio in relation in chromosomes?

Answer 22

The packing ratio is the length of the DNA to the unit length of the fibre containing it. In other words, it is the degree of condensation of the DNA.

• Ex. Shortest human chromosome (with 4.6 x 10^7 bp of DNA) has an extended length of 14, 000 micrometer and a condensed length of 2 micrometer The packing ratio of this is 7000 (14,000/2)

Question 23

What is the nucleosome?

Answer 23

The nucleosome is a subunit of chromatin. Each nucleosome is composed of eight histone proteins as well as the DNA that is wrapped around them.

Question 24

What does the nucleosomal DNA consist of?

Answer 24

1. Core DNA
2. Linker DNA

Question 25

What is the core DNA in the nucleosome?

Answer 25

The core DNA is 147 bp in length and wraps around 1.65 times on the outside of the histone October (8 histone proteins). This DNA is a feature present in all eukaryotes and this type of DNA is resistant to Micrococcal nuclease (Mnase).

Question 26

What is Micrococcal Nuclease?

Answer 26

A enzyme that trims and digests the nucleosomes to core particles. Core DNA in nucleosomes is resistant to this enzyme.

Question 27

What is linker DNA in nucleosomes?

Answer 27

Linker DNA is the DNA that is present between each nucleosome. The length of this type of DNA is between 13-110 bp.

Question 28

What are histones? Name the 5 abundant histones present in eukaryotic cells.

Answer 28

Histones are small basic proteins (rich in K and R residue) which have a high affinity for DNA.
The 5 abundant histones in eukaryotic cells include H1, H2A, H2B, H3, and H4.

Question 29

What histones are part of the histone octamer?

Answer 29

Two copies of H2A, H2B, H3, H4 (These are known as the core histones).

Question 30

Why is the H1 Histone not apart of the histone octamer?

Answer 30

Because it is associated with linker DNA and needs to be at the point where DNA enters or exits the nucleosome. The central globular region of the H1 histone is what connects to the histone octamer.

Question 31

How does nucleosome assembly occur?

Answer 31

1. First, the H3 and H4 tetramer binds to the DNA
2. Next, the H2A and H2B dimers join the H3/H4 - DNA complex to form the nucleosome

Question 32

How are non-histone proteins associated with chromosomes?

Answer 32

Non-histone proteins such as DNA binding proteins help with regulating replication, repair, recombination, and transcription of the cellular DNA.

Question 33

What is the histone-fold domain?

Answer 33

The histone-fold domain is composed of 3 alpha-helical regions which are separated by 2 unstructured loops. Each of the core proteins (H2A, H2B, H3, H4) have the histone-fold domain.

Question 34

What is the purpose of the histone-fold domain?

Answer 34

The histone-fold domain helps to mediate the formation of the head to tail heterodimers for specific pairs of histones.

Question 35

What is the purpose of the N-terminal tails on the histone-fold domain?

Answer 35

1. To stabilize DNA wrapping around the octamer by forming hydrogen bonds with the DNA
2. Serves as sites for extensive post-translational modifications

Question 36

What are the two structurally and functionally distinguishable territories within chromosomes?

Answer 36

1. Heterochromatin
2. Euchromatin

(Slide 22 on lecture 1 to learn more about each of these)

Question 37

What is the primary structure of chromatin?

Answer 37

10-11 nm fibre which consists of a string of nucleosomes which have a packing ratio go 6

Question 38

What is the secondary structure of chromatin?

Answer 38

30 nm fibres which contain 6 nucleosomes/turn, this is due to the interactions between neighbouring nucleosomes which leads to the formation of more condensed fibres. The fibres are organized into either a one-start solenoid or a two-start zig-zag which has a packing ratio of 40.

Question 39

What helps to promote the formation of secondary structures in chromatin organization?

Answer 39

• Histone protein H1
• Histone tails
• Increased ionic strength

Question 40

How does the formation of the 30 nm fibre occur in chromatin organization?

Answer 40

The H1 histone binds to the nucleosome at the linker DNA and at the middle of the 147 bp core DNA. Due to this binding, a defined angle is formed for DNA entry and exit from the nucleosome leading to the zig-zag appearance of the nucleosomal DNA.
Binding of the H1 histone also helps to stabilize the higher order chromatin structures which helps the nucleosomal DNA to form the 30 nm fibre

Question 41

Describe the one-starter solenoid model for secondary chromatin structure.

Answer 41

The nucleosomal DNA forms a super helix which contains ~ 6 nucleosomes per turn and the linker DNA is buried in the centre of the helix. The packing ratio is 40.

Question 42

Describe the zig zag model.

Answer 42

30 nm fibre is a compacted form of zig-zag nucleosome arrays. The linker DNA passes through the central axis of the fibre and therefore, longer linker DNA favours the zig-zag model.

Question 43

What the nuclear scaffold?

Answer 43

In order to form fully compact DNA, more folding is required. There is a theory on the folding structure of the DNA which proposes that the 30 nm fibre forms 40-90 kb which are held together at their bases by the nuclear scaffold. A protein complex in which the 30 nm fibre wounds around.

Question 44

What classes of proteins contribute to the nuclear scaffold?

Answer 44

• Topoisomerase II which is involved in the supercoiling of the DNA and is abundant in both the nuclear scaffold and mitotic chromosomes
• SMC proteins which help with the structural maintenance of the chromosomes by condensing and holding sister chromatids together after chromosome duplication.

Question 45

What is the final level for chromatin organization and chromosome packaging?

Answer 45

In this level, the DNA is packaged into large loops of the 30 nm fibre which are held to the nuclear scaffold at their base. ( Image on slides 29 + 30 on Lecture 1)