Lecture theme 1 Levels of gene control

Question 1

How to demonstrate gene control.

Answer 1

 Strategies for detecting mRNA expression Northern blotting, in situ hybridization, RT/PCR, micro-array, RNA-seq

 Strategies for detecting protein expression PAGE, 2D PAGE, Western blotting, in situ antibody detection, liquid chromatography

 Strategies for studying control on DNA level Southern blotting, DNA sequencing

Question 2

Detecting a single protein: Western blotting.

Answer 2

• separate the protein in tissues A and B by SDS
• blot protein to a membrane
• use protein-specific antibodies coupled to a marker to detect.
In this case the casein kinase protein.

Question 3

1-Detecting overall protein content: two dimensional PAGE.

2-How do we determine the significance of the protein spots observed by 2D PAGE?

Answer 3

1)
 1 st dim. iso-eletrofocusing (differences in charge)
 2 nd dim. SDS polyacrylamide gel electrophoresis (size)

Proteins present at similar levels in both tissue.
Proteins present in only one tissue.
Proteins present in in both tissue but at different levels.

2)
A) excise the spot of interest and digest it with trypsin
B) individual peptides
Obtain molecular weight of peptide by MALDI.
Obtain sequence of peptide by nanoelectrospray mass spectrometry.
C) search the data base to see from which protein the peptide is derived.

Question 4

To detect single gene-specific mRNA: Northern blotting

- in this case α-fetoprotein mRNA

Answer 4

• isolate total mRNA from cell types A and
• separate mRNAs by electrophoresis
• Transfer mRNA to nylon membrane
• label gene-specific probe to detect mRNA by hybridization

Question 5

To detect specific mRNA: Quantitative reverse transcriptase polymerase chain reaction (RT/PCR).

Answer 5

1. mRNA first converted to a complementary copy (cDNA) by the enzyme reverse transcriptase
2. primers that hybridize specifically to the cDNA are then used in a PCR for amplification.

Method allows for:

1. detection of very low levels of mRNA expression
2. precise quantification of amounts of mRNA e.g. can analyse different tissues, time intervals, drug treatments, etc.

Question 6

To analyse total mRNA profile: microarray (gene-chip).

Answer 6

1. spot cDNA clones representing all possible genes on a chip
2. isolate mRNA from cell or tissue, label with fluorescent marker; hybridize to cDNA on chip
3. powerful method to compare expression profiles of tissues in response to stimuli / treatments

Question 7

To study individual genes: Southern blotting.

Answer 7

 isolate DNA from tissue, digest with restriction enzyme(s), gel electrophoresis

 transfer to membrane

 hybridize with gene-specific probe

Question 8

Proof that differential expression is not due to DNA loss.

Answer 8

1-DNA loss as a mechanism of gene control in rare cases

• Red blood cell differentiation: involves complete loss of cell nucleus i.e. whole genome
- highly specialised cell; synthesizes large amounts of globin for oxygen transport

2-DNA amplification as a mechanism of gene control
• Amplification of certain genes in certain tissues, eg germline
• In highly selective cases when genes need to be expressed at very high levels in short period of time
- eg chorion genes in embryonic development of Drosophila

3-DNA rearrangement as a mechanism of control Genes are linked in a new order and unwanted intergenic regions removed.
 only in specialised cases, eg synthesis of antibodies

Question 9

Transcriptional vs post-transcriptional control

How to distinguish between transcriptional or post- transcriptional control? Studies of nuclear RNA

Answer 9

If gene A is regulated at transcriptional level:
 levels of primary mRNA in nucleus will correspond to levels of mature mRNA in cytoplasm
 larger unspliced precursors of gene A may be present in nucleus (still unprocessed)

If gene A is regulated at post- transcript. level:
 smaller mRNAs may be present in nucleus due to mRNA degradation levels of primary mRNA in nucleus will differ from levels of mature mRNA in cytoplasm.

Strategy: Extract a nuclear RNA fraction as well as a cytoplasmic RNA fraction from cells of interest, analyse by Northern blotting
 evaluate results to obtain answer about which level of regulation.

Question 10

Techniques to study the rate of mRNA synthesis.

Answer 10

1-Pulse labeling assay
 measures the incorporation of radioactive NTPs into newly synthesized transcripts
 radioactive pulse performed within cell
 results may give indication of mRNA stability

2-Nuclear run-on transcription assay
 similar to above, but radioactive pulse performed within isolated nuclei
 strong evidence for control at level of transcription initiation
 method not influenced by mRNA stability

3-Quantitative Reverse Transcriptase PCR (qRT/PCR)
Very sensitive
allows analyses of genes that are transcribed at very low levels very precise quantification

Question 11

1-Pulse labeling assay.

2-Nuclear run-on transcription assay

Answer 11

1)
Add radioactively labelled nucleotides to cells.
After 5-10min harvest cells and isolate RNA
Hybridize labelled RNA to dot blot containing DNA from genes the transcription of which is being measured.

2
isolate the nuclei from the cells.
Add radioactively labelled nucleotides to the isolated nuclei.
After 1-2h isolate RNA from nuclei
Hybridize labelled RNA to dot blot containing DNA from genes the transcription of which is being measured.

Question 12

Genes are controlled at different levels.

Answer 12

 Genome level
- number of gene copies available

 Transcription level
- number of mRNAs copies transcribed per gene

 Post-transcriptional level
- number of stable mRNAs copies in the cytoplasm

 Translation level
- number of proteins translated per mRNA copy

 Post-translational level
- number of protein copies modified after translation