0514 - Gene expression and regulation - AK

Question 1

What is Gene Expression?

Answer 1

Gene Expression is the process of synthesizing a functional gene product from DNA. These products are often proteins (coded from Exons), but the products of introns can include functional RNA (e.g. ribosomal RNA, transfer RNA and small Nuclear RNA).

Question 2

What is Gene Regulation?

Answer 2

Gene regulation is any one or combination of processes that leads to a change in the natural level of expression of a gene.

Question 3

At what level is Gene Expression Regulated?

Answer 3

They may be regulated at the level of Transcriptional Regulation (e.g. Transcription Factors), Post-Transcriptional Regulation or Epigenetics (e.g. DNA Methylation).

Question 4

How can Transcription Factors affect gene expression?

Answer 4

Transcription Factors may activate (turn on a gene that was previously turned ‘off’), enhance (increase the activity of an already active gene), repress (dull the activity of a gene) or silence (turn a gene ‘off’) gene expression.

Question 5

What is a Transcription Factor?

Answer 5

Transcription Factors are a group of proteins that recognize and a bind a short DNA sequence at the Promoter and/or Regulatory Sequences, varying the expression of a gene (stimulatory or inhibitory effect).

Question 6

What is a TATA Box?

Answer 6

A TATA Box is a homologous sequence (5’–>3’; TATAAA) found in most promoters, and are regulated by a subset of Transcription Factors, known as General Transcription Factors (GTF).

Question 7

What is NF-KB?

Answer 7

Nuclear Factor Kappa Light-Chain Enhancer of Activated B-Cells is a Transcription Factor crucial in initiating an immune response to infection by enhancing cytokine production for cell survival.

Question 8

How is NF-KB regulated?

Answer 8

NF-KB is regulated by IKB-alpha, which is constitutively bound to repress NF-KB’s activity. Upon Ligand binding (e.g. TNF-alpha), IKB-alpha is phosphorylated by IKK (a kinase). Phosphorylation of IKB-alpha causes the dissociation of IKB-alpha from NF-KB and targets IKB-alpha for proteosomal degradation. The dissociation from IKB-Alpha allows NF-KB to traverse into the Nucleus to bind to the promoters of pro-inflammatory cytokine and increase their transcription.

Question 9

What is Phosphorylation?

Answer 9

Phosphorylation is the addition of a Phosphate group via Kinase to a protein, which causes a conformational change in the protein, resulting in a change in activity.

Question 10

What are the effects of Phosphorylation?

Answer 10

Phosphorylation can either make a protein more active, inactive, inhibit its action, enhance its actions, or target the protein for degradation.

Question 11

What is Apoptosis?

Answer 11

Apoptosis is the process of programmed cell-death, which features shrinkage and fragmentation of cells and nuclei, and degradation of the Chromosomal DNA.

Question 12

What can induce Apoptosis?

Answer 12

Heat, Radiation, Nutrient Depravation, Viral Infection, Hypoxia, Hyperoxia and Membrane Damage.

Question 13

What is Alternative Splicing?

Answer 13

Alternative Splicing is a regulated process during gene expression that results in a single gene coding Multiple Proteins. The pre-mRNA is spliced into multiple different isoforms leading to the creation of different proteins with different biological functions. E.g. presence of an Exon which codes for a phosphorylation would enable the protein to have kinase-activity.

Question 14

What is PCR?

Answer 14

Polymerase Chain Reaction (PCR) is a technique used to amplify a specific segment of DNA between two regions of a known sequences from a complex DNA mixture. PCR allows for genetic testing, tissue typing (transplantation), infectious disease diagnosis, forensics and research.

Question 15

What are the general steps to a PCR?

Answer 15

1. Denaturation of DNA 2. Annealing Temperature3. Extension

Question 16

What is Quantitative PCR?

Answer 16

Quantitative PCR (qPCR) is conducted in a similar way to PCR, except that qPCR uses a fluorescent reporter (SYBR Green), which detects an increase in DNA product.

Question 17

How do you interpret qPCR results?

Answer 17

There are two things to look for on a qPCR graph. You would examine to see if there is a peak in fluorescence, as the intensity of fluorescence correlates to amount of DNA. Secondly, you would note the cycle number at which this peak occurred, as this may be indicative of increased endogenous gene expression. The lower the cycle number for which this peak is reached, the higher amount of DNA was already present and thus less time was needed to reach critical ‘fluorescence’ concentration.