Biology 3594A Study Guide - Final Guide: Virus Quantification, Lambda Phage, Blue Budgerigar Mutation
13 May 2018
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Lecture 9 Flashcards
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Select-cII Big Blue in vivo mutation report assay
- what it measures
big blue = mutations made a mutant phenotype wtih
blue plaque
- assay to find the mutants (blue plaque)
- harvest DNA from mouse and see if viral genome
has mutations (then could sequence if you want)
- mutations arise in vivo but mutant phenotype is
detected in vitro
Select assay vs screening assay
In select assay, only the mutant appears with
specific temperature condition
Screening assay: looking at all cII genes and finding
the mutant ones
Transgenic mouse
Mouse genome contains an integratedlambda phage
genome with a cII gene that controls lysis of
bacterialhost and plaque formation
- lambda phage infects E coli
- infected E coli create a plaque
Select-cII Big Blue in vivo mutation report assay
- process
1. Buy transgenic mouse with a mutation reporter
gene
2. Harvest Bigblue bone marrow
3. Extract DNA
4. Add protein capsid, so some phage is packaged.
5. Phage then infects E coli
6. E coli plated at 2 temperatures
- Incubate at 24 C, only mutant cII phage lyse
bacteria
- Incubate at 37 C, all phage lyse bacteria
Compare the plates
7. collect mutants off plate, isolate DNA, and
sequence cII gene to identify the mutations
Mutant frequency
Total mutants found (24 C) / total number of
mutants you would see out of a mouse (37 C)
No. of mutants with mutations / total no. of plaques
Mutant cII genes / all cII genes harvested
find more resources at oneclass.com
find more resources at oneclass.com
allows you to tell how potent something is as a
mutagen
Mutation pattern
Relative number of different types of mutations
Mutant
describes a phenotype
ENU
ethylnitrosourea
- potent germline mutagen (affects sperm and egg)
- used in mutation screening assays to inactivate
genes and study gene function
- alkylating agents
- transfers ethyl group to thymine
- used to test to make sure big Blue system was
working
elevated endogenous acetaldehyde
- effect on mutant frequency
No significant change in mutant frequency
- surprising, they demonstrated a lot of DNA damage
so why aren't there mutations?
- no change when elevating acetaldehyde and
knocking out repair... why?
Experiment: elevation of endogenous
acetaldehyde increases the number of
substitutions per genome
- extract bone marrow and HSC from donor mouse
that was subjected to high levels of acetaldehyde -
take individual HSCs and grow them up into clones
- give them to another recipient mouse that can
populate the bone marrow
- after 16 weeks extract the cells and see if an
individual cell has more mutations
- they got the expected result!
- elevated levels of acetaldehyde + KO repair
mechanisms = more substitutions
- but had to look in a single cell to get it "per
genome"
Mutation landscapes
Different types of mutationsplotted along the mouse
DNA sequence
- gives you position in the genome
- can see where mutations occur
Circos plots
see a whole genome and its mutation landscape
- can see more substitutes per genome in Aldh2-/-
Fancd2-/- HSCs vs wild type HSCs
Endogenous aldehydes mutate the HSC genome
- abundance of types of mutations
More de novo deletions than insertions
- de novo small deletions
- microhomology-mediated deletions
Microhomology
Small regions of similarity because of a common
ancestor
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Select-cii big blue in vivo mutation report assay. Mutant frequency big blue = mutations made a mutant phenotype wtih blue plaque. Assay to find the mutants (blue plaque) Harvest dna from mouse and see if viral genome has mutations (then could sequence if you want) Mutations arise in vivo but mutant phenotype is detected in vitro. In select assay, only the mutant appears with specific temperature condition. Screening assay: looking at all cii genes and finding the mutant ones. Mouse genome contains an integratedlambda phage genome with a cii gene that controls lysis of bacterialhost and plaque formation. Incubate at 24 c, only mutant cii phage lyse bacteria. Incubate at 37 c, all phage lyse bacteria. Compare the plates: collect mutants off plate, isolate dna, and sequence cii gene to identify the mutations. Total mutants found (24 c) / total number of mutants you would see out of a mouse (37 c)
