Organisation and control of eukaryotic genome

Question 1

Histone modification

Answer 1

1. Histone acetylation involves the covalent addition of acetyl groups to positively-charged lysine residues in the tails of histone proteins. The positive charge is neutralised and does not bind to negatively charged DNA
2. Acetylation reduces interaction between the histones and DNA, making it less compact and more accessible to the transcriptional machinery for the initiation of transcription
3. Histone acetyltransferase (HATs) catalyses the addition of acetyl groups and promote transcription
4. Histone deacetylases (HDACs) catalyses the removal of an acetyl group and inhibit transcription

Question 2

DNA modification

Answer 2

1. The addition of methyl groups on certain bases (usually cytosine) of DNA is a common method of DNA silencing
2. The transfer of a methyl group to the cytosine is catalysed by DNA methyltransferase
3. Methylated DNA attracts other proteins which and in turn recruits histone deacetylation enzymes
4. Association of these proteins and HDACs makes the DNA more compact and inhibits transcription

Question 3

Transcriptional control

Answer 3

In eukaryotes, genes are transcribed at the appropriate time, in the appropriate cell type and in the the appropriate amounts , and can be regulated

by

• enhancers and silencers

Question 4

Post-transcriptional control: RNA processing

Answer 4

1. By 5’ Capping, addition of 3’ Poly-A tail and splicing of mRNA
2. Alternative splicing also occurs to regulate gene expression.
   It is the RNA splicing variation mechanism in which the exons of the pre-mRNA are separated and ligated so as to produce alternative mature mRNA arrangements
   The significance is that many different mRNA molecules and polypeptides can be made from a single gene. It increases the number of proteins without increasing the number of genes in a genome

Question 5

Post-transcriptional control: RNA transport

Answer 5

1. Eukaryotes have a nuclear membrane which prevents simultaneous transcription and translation. This serves as a form of control of gene expression
2. Unprocessed RNA or incompletely processed RNA is degraded in the nucleus and the gene is effectively not expressed.
   Therefore, COGE can be achieved by controlling transport of RNA out of nucleus

Question 6

Translational control: Translation initiation

Answer 6

1. Initiation of translation requires translation initiation factors. Phosphorylation and dephosphorylation can activate or inactivate translation initiation factors needed to initiate ribosomal binding

Question 7

Translational control: Translation repressors

Answer 7

1. Translational repressors are proteins which bind to mRNA. These regulatory proteins bind at the 5’ UTR, preventing initiation or prevent ribosome from progressing

Question 8

Translational control: Stability of mRNA (Half-live of mRNA)

Answer 8

1. The more stable the mRNA molecule is, the longer it is translated and more polypeptides are produced

Question 9

Translational control: Cytoplasmic polyadenylation

Answer 9

1. Cytoplasmic polymerase catalyse the cytoplasmic polyadenylation of the 3’ end, and this activates translation of mRNA into polypeptide
2. This allows temporal control of when exactly a polypeptide to be translated is regulated

Question 10

Translational control: Localisation of mRNA

Answer 10

1. RNA localisation has been observed in many organisms and this is likely a common mechanism for cells to concentrate high mRNA levels to ensure a high level of polypeptide to be synthesized at a specific location where the polypeptide is needed most
   This allows spatial control of gene expression

Question 11

Post-translational control: biochemical modification

Answer 11

1. By attaching other biochemical functional groups such as lipids, carbohydrates and phosphate groups to the protein, the protein is activated or becomes functional for its role in the cell

Question 12

Post-translational control: structural modification

Answer 12

1. Structural modification such as removing amino acids from the protein may occur to produce the mature protein

Question 13

Post-translational control: Protein degradation

Answer 13

1. Excess or misfolded proteins can be degraded by proteasomes. it offers a mechanism whereby specific proteins can be quickly targeted and degraded
2. Small proteins called ubiquitin are covalently attached to unwanted proteins
3. Proteasome recognises and binds to these ubiquitin-tagged proteins
4. Proteasome unfolds the tagged protein and injects it into the core of the proteasome while ubiquitin is released during the entry
5. Within the core, the protein is degraded into short peptides or amino acids which are recycled into the cytoplasm

Question 14

Post-transcriptional control

Answer 14

• RNA processing
• RNA transport

Question 15

Translational control

Answer 15

• Translation initiation
• Translation repressors
• Stability of mRNA (Half-life of mRNA)
• Cytoplasmic adenylation
• Localisation of mRNA

Question 16

Post-translational control

Answer 16

• Biochemical modification
• Structural modification
• Protein degradation

Question 17

Polymerase chain reaction (PCR) and its components

Answer 17

• is a process used to amplify a specific segment of DNA in vitro. It results in synthesis of large amounts of DNA from a minute amount of starting material
• DNA polymerase such as tag polymerase is used. It is a thermostable DNA polymerase isolated from the bacterium.
• DNA primers are single-stranded DNA molecules. 2 different DNA primers complementary to the flanking sequence of target DNA sequence to be amplified are needed
• Deoxyribonucleotides are substrates for DNA replication in PCR

Question 18

Process of PCR

Answer 18

1. Denaturation
   - heat DNA to a high temperature of about 95 degree Celsius
   - Denaturation of double-stranded DNA by breaking hydrogen bonds between nitrogenous bases of the 2 strands
2. Annealing
   - temperature is lowered to about 50-60 degree Celsius
   - primers anneal via hydrogen bonding to the flanking sequence of the target DNA sequence. It binds due to complementary base pairing to the single-stranded DNA
3. Elongation
   - temperature is increases to about 72 degree Celsius for chain elongation
   - tag polymerase adds nucleotides to the 3’OH end of the primers using the DNA molecule as a template

*this 3 processes are repeated many times. This is called a chain reaction as the products of the previous reaction are used as reactants in the next cycle. Amount of target DNA sequence increases exponentially

Question 19

Advantages of PCR

Answer 19

1. Large amounts of DNA can be produced from very minute amounts of starting materials
2. Large amounts of DNA can be produced in a short period of time with relatively high accuracy of replication
3. Specific sequences of DNA can be amplified but using specific primers

Question 20

Limitations of PCR

Answer 20

1. Knowledge of the DNA or amino acid sequences of target gene or protein is required to Synthesise flanking nucleotide primers
2. Primers are short nucleotide sequences so non-target DNA sequences may be amplified
3. Tag polymerase does not perform proofreading so there may be mistakes in complementarity of the nucleotides added and theses mistakes may be amplified
4. There are limits to the length of target DNA sequence to be amplified

Question 21

Gel electrophoresis

Answer 21

• technique of separating nucleic acids or proteins based on the size, electrical charge and other physical properties of the molecule by passing them through a gel
• the rate at which a macromolecule travels is inversely proportional to its size

1. The agarose gel is prepared by heating agar powder with buffer solution to dissolve it.
2. The agarose gel solution is then poured into a gel tray and cooled. A comb is added at one end of the gel to create the wells for the loading of the DNA
3. After the agarose has cooled and hardened, the comb is removed to reveal the wells. The gel is then placed within an electrophoresis chamber filled with buffer solution
4. DNA samples are then loaded into the wells . The gel is orientated such that the wells are placed at the negative electrode. Small amount of loading dye is mixed with sample.
   - The blue dye will move along the gel band give an indication of the progress of electrophoresis.
   - Glycerol is usually added to loading dye as it is dense, allowing DNA to sink into the wells.
   - in most cases, a standard DNA ladder is also loaded so that the size of DNA fragments can be estimated
5. The direct current is turned on. After the DNA has run to about 2/3 of the gel length, current is stopped
6. Ethidium bromide is added to the buffer or the agarose gel before cooling. When viewed under UV radiation, DNA bands fluoresce

Question 22

Nucleic acid hybridisation

Answer 22

• is the cbp of a nucleic acid to another nucleic acid to form a double stranded molecule. Carried out using a nucleic acid probe. DNA probe is a single-stranded DNA sequence that is complementary to the gene of interest and is usually labelled

Question 23

Southern blotting

Answer 23

• a method that combines gel electrophoresis and nucleic acid hybridisation. it involves a transfer of separated DNA fragments onto a membrane for hybridisation

1. DNA samples are purified and cut with restriction enzymes
2. DNA fragments are separated using gel electrophoresis
3. Double-stranded DNA is denatured into single strands with addition of sodium hydroxide. Hydrogen bonds between complementary bases are broken
4. Bands on the gel are transferred to a nitrocellulose membrane via capillary action.
   - the gel is placed in a basin of buffer solution containing sodium hydroxide . A piece of positively-charged nitrocellulose membrane is paced directly above the gel
   - large amounts of dry paper towels and heavy wrights are then placed on top of the setup to generate capillary action. The dry paper towels absorb buffer solution through the gel and membrane
   - capillary action will transfer the single-stranded DNA bands onto the nitrocellulose membrane corresponding to those on the gel
5. Negatively charged DNA forms temporary bonds with the positively charged membrane. The nitrocellulose membrane is then subjected to high temp or UV light to permanently cross link single stranded DNA to the membrane
6. Nitrocellulose membrane containing the single-stranded DNA is incubated in the solution containing the labelled DNA probe.
7. Depending on what the probe is labelled with, different methods of detection is used. In this case, DNA probe is labelled with nucleotides containing the radioactive 32P.
8. the probe binds to the complementary DNA fragment of interest via hydrogen bonds
9. When exposed to a piece of X ray film, the radioactive isotope exposes the film.
10. Colorimetric methods involving a DNA probe labelled with luminescence molecule