Molecular Biology 2

Question 1

How many nucleotide pairs in the humane genome ?

Answer 1

The human genome contains about 3 billion nucleotide pairs organized as 23 chromosome pairs.

Question 2

How long would the DNA of each chromosome measure w/o any packaging ?
How big is the nucleus ?

Answer 2

The DNA contained by each of the chromosomes would measure between 17 and 85 mm long.
Yet the nucleus is about 0.006 mm in length.

Question 3

What is the packaging ratio ?

Answer 3

Length of B-form DNA / actual length

Question 4

What is the cytological difference between euchromatin and heterochromatin ?
What do these differences reflect ?

Answer 4

Originally, the two forms were distinguished cytologically by how intensely they stained : the euchromatin is less intense, while heterochromatin stains intensely, indicating tighter packing.
These 2 forms reflect the level of activity of the cell. Euchromatin is prevalent in cells that are active in the transcription of many of their genes whileheterochromatin is most abundant in cells that are less active or not active.

Question 5

What are the 8 steps of DNA packaging ?

Answer 5

1. At the simplest level, chromatin is a double stranded helical structure of DNA.
2. DNA is complexed with histones to form nucleosomes.
3. Each nucleosome consists of 8 histone proteins around which the DNA wraps 1.65 times.
4. A chromatosome consists of a nucleosome plus the H1 histone.
5. The nucleosomes fold up to produce a 30-nm fiber …
6. … that forms loops averaging 300nm in length.
7. The 300-nm fibers are compressed and folded to produce a 250-nm-wide fiber.
8. Tight coiling of the 250-nm fiber produces the chromatid of a chromosome.

Question 6

What are the 2 amino acids that are present in very large quantities in histones ?
What property does this give to histones ?

Answer 6

Between 20% and 32% of lysine and arginine –> gves histones a +ve charge

Question 7

What are the different types of histones ?
What are their MW ?
What id their proportion of lysine and arginine ?

Answer 7

Core histones :
- H2A : 14,000 Da, 20% arg/lys
- H2B : 13.900 Da, 22% arg/lys
- H3 : 15,400 Da, 23% arg/lys
- H4 : 11,400 Da, 24% arg/lys
Linker histone :
- H1 : 20,800 Da, 32% arg/lys

Question 8

What is a histone octamer made of ?

Answer 8

Core histone (2 of each –> 2*4=8)

Question 9

How is the histone octamer formed ?

Answer 9

H3+H4 = H3-H4 dimer
H2A+H2B = H2A-H2B dimer
H3-H4 tetramer + H2A-H2B tetramer = histone octamer

Question 10

What is a nucleosome ?

What is it made of ?

Answer 10

The nucleosome is the basic repeating unit of the 11 nm chromatin fibre.

• 11 nm in diameter, 100 kD
• DNA double helix wraps twice around a central core of a histone octamer
• histone H1 fasterns the DNA to the nucleosome core

Question 11

How long (in bp) is the DNA strand wrapped around a single histone core ?
What about the regions in between histones ?

Answer 11

Core DNA = 146bp

Linker DNA = 20-60 bp

Question 12

How is the nucleosome fiber formed ?

Answer 12

Nucleosomes are usually packed together, with the aid of a histone H1, to form a 30nm fibre.

Question 13

How big is :

• the metaphase chromosome ?
• the condensed scaffold-associated chromatin ?
• the extended scaffold-associated chromatin (during interphase) ?
• a 30-nm chromatin fiber of packed nucleosomes ?
• the “beads on a string” form of chromatin ?
• the DNA double helix ?

Answer 13

Metaphase chromosome = 1400 nm
Condensed scaffold-associated chromatin : 700 nm
Extended scaffold-associated chromatin (during interphase) = 300nm
30-nm chromatin fiber of packed nucleosomes = 30 nm
“beads on a string” form of chromatin = 11 nm
DNA double helix = 2 nm

Question 14

What is the packaging ratio of interphase chromosome ?

What about mitotic chromosomes ?

Answer 14

Interphase chromosomes : 1,000

Mitotic chromosomes : 10,000

Question 15

How is the condensation of the nucleosome regulated ?

Answer 15

Histone tails are modified and this alters the condensation of the nucleosome.
Modifications include methylation and acetylation.

Question 16

What is the specificity of condensed chromatin ?

Answer 16

It is less accessible.

Question 17

What interactions/forces contribute to DNA stability ?

Answer 17

Hydrogen Bonding - provides specificity as well as overall stability
Stacking - induced dipole interactions between aromatic bases
Presence of cations (metal cations and cationic proteins) - counteracts electrostatic repulsion of anionic phosphate backbone
Hydrophobic effects - burying the hydrophobic purine and pyramid ring in the interior of the double helix increases its stability

Question 18

How can DNA be denatured ?

Answer 18

Heat of chemicals (high pH)

Question 19

What is the overall charge of DNA ?

Answer 19

-ve

Question 20

What are the features of DNA and RNA ?

Answer 20

Both capable of re-annealing, susceptible to enzymes, etc.

Question 21

At what temperature does DNA melt ?

What does the specific DNA melting temperature depend on ?

Answer 21

Approx 70-100 degress Celsius

DNA melting temperature depends of sequence composition

Question 22

After 2 strands of DNA have been seperated, what happens upon cooling ?
Why is this important ?

Answer 22

• Upon cooling the two strands will pair back and renature the duplex in a process called REANNEALING.
• The ability of single strands of DNA (or RNA) to pair with its exact complementary strand is the most important property of nucleic acids.
• Complementary pairing is crucial for the physiological role of DNA and lies at the heart of several important molecular biology techniques.

Question 23

How can the specific base pairing of DNA be exploited ?

Answer 23

DNA re-annealing and hybridization.

Question 24

What are common enzymes used for DNA engineering ?

Answer 24

Polymerases (DNA polymerases, reverse transcriptase) Nucleases (e.g. restriction endonucleases, RNAse H) DNA ligase
Alkaline Phosphatase

Question 25

What is gel electrophoresis ?

Answer 25

Gel electrophoresis is a method used to separate mixtures of DNA, RNA, or proteins according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores. The molecules travel through the pores in the gel at a speed that is inversely related to their lengths. The bands can be viewed by staining w/ EtBr and exposing the gel to UV light.

Question 26

What is the “probe” in southern blotting ?

Answer 26

A nucleic acid (usually DNA) that is complementary to specific gene or nucleic acid sequence of interest.

Question 27

What is the stringency of hybridization ?

Answer 27

High stringency = base pairing between highly similar sequences
Low stringency = base pairing between less similar sequences

Question 28

What is a DNA microarray ?

What is it used for ?

Answer 28

A DNA microarray (DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles (10^−12 moles) of a specific DNA sequence, known as probes (or reporters or oligos). These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample (called target) under high-stringency conditions.

Question 29

What is FISH ?

Answer 29

Fluorescent In Situ Hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity.