MBIO 4410 Lecture 2:
21 Jan 2019
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MBIO 4410
Lecture 2
Detect and measure macromolecular synthesis
- How do you know what comes in vs. what was already inside?
o Need a method of tagging what's being made
o Most are bulky tags and can alter what is being synthesized.
o Using isotopic labels is easily differentiated and doesn’t affect function - but can
be dangerous
o Label the precursors such as amino acids so you can see if they're being
incorporated into proteins.
o Differentiate between precursor and final product
- How to detect and measure macromolecular synthesis? (NOTE: don’t memorize the
steps, just need to differentiate between newly synthesized biomolecules vs. pre-
existing)
o Differentiation of precursors from synthesized macromolecules done by
differential precipitation
▪ Ie. Smaller sizes come out and larger ones stay in so you can see what the
precursors are
o Thus, pre-label cells with radio-labeled precursors for DNA, RNA and protein
o Infect cells
o Harvest cells at variety of times post-infection
o Chill to stop macromolecular synthesis, lyse cells
o Precipitate with 10% trichloracetic acid
o Filter each time point through coarse glass fibre filters
o Count each filter in scintillation counter
The Baltimore Classification Scheme
- Class I
o Ex. Herpesvirus, Adenovirus
o Is a double stranded DNA virus, that makes mRNA and completes DNA
replication using the DNA as the template
o Kinetics graph would show RNA, protein and then DNA
- Class II
o Ex. Parvovirus
o Is a single stranded DNA virus, either positive or negative DNA strand
o First the virus needs to make the ssDNA into dsDNA using host proteins, then
makes mRNA, proteins and finally replicates at the end
▪ The genome itself doesn’t get replicated until viral proteins are made
• Polymerases
• Cofactors
• Most virus use host enzymes for DNA replication
- Class III
o Ex. Reoviruses
Document Summary
How to detect and measure macromolecular synthesis? (note: don"t memorize the steps, just need to differentiate between newly synthesized biomolecules vs. pre- existing: differentiation of precursors from synthesized macromolecules done by differential precipitation. Smaller sizes come out and larger ones stay in so you can see what the precursors are: thus, pre-label cells with radio-labeled precursors for dna, rna and protein. Infect cells: harvest cells at variety of times post-infection, chill to stop macromolecular synthesis, lyse cells, precipitate with 10% trichloracetic acid, filter each time point through coarse glass fibre filters, count each filter in scintillation counter. Is a double stranded dna virus, that makes mrna and completes dna replication using the dna as the template: kinetics graph would show rna, protein and then dna. Poliovirus, west nile, rubella, tmv: from the mrna the rna strand is made which is used for rna replication to.
