![Page 1: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/1.jpg)
APOG
![Page 2: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/2.jpg)
Today’s schedule:
• SPIN• How genetic dissection works• Where we are and where we are going• Reminder about proposal• Mutagenesis and Screens• Analyzing mutants• Introduction to C. elegans• I will post the notes after class
![Page 3: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/3.jpg)
• The study of processes that are the domain of genetics: DNA and information storage, replication, transmission. (done)
• The methodology of genetics as a system of logic for studying any biological process.
(We are here now.)
Genetics
![Page 4: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/4.jpg)
Frans’s rules of genetics
• An EMS mutagenesis is the most powerful functional genomics tool.
• The exceptions are more interesting than the ones that follow the rules.
• Many genetic operations are self-referential, that is you continue to build an argument or case based on a preponderance of evidence.
• The logic of genetics (removing a gene at a time in vivo) stands by itself-– but because we can we will confirm genetic results with molecular
biology and biochemistry.
• Genetics can teach us fundamental properties of evolution.
![Page 5: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/5.jpg)
Five more miscellaneous corollaries:
• Every gene has a function.• Genetic background matters.• Genome sequences are our second most valuable
tool (next to mutagens)(These are the anatomy for geneticists)
• Genetics versus Sciteneg• Genetics is the best science for a biologist because
of all of the above reasons and because of its rigor.
![Page 6: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/6.jpg)
Goals of genetic analysis
Identify components
Assign roles
Establish hierarchy
Build on the hierarchy
![Page 7: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/7.jpg)
How?
• Components: mutagenesis, genetic screens for mutations with a particular phenotype
• Assign roles: genetic complementation test, test for whether alleles are dominant or recessive and nulls
• Hierarchy: epistasis analysis, other genetic interactions
• Establish molecular pathway using forward and reverse genetic tools, cell biology, biochemistry, etc.
![Page 8: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/8.jpg)
Logic and Rationale
Comprehensive: all componentsSystematic: identify genes and understand their rolesPrecise: mutate one component at a timePowerful: remove one and only one component (and observe the consequences for functionCertain: if approach is systematic and biology
permitsValid: Intrinsic logic-examine the roles of genes
and how they relate to one another,self-referential
![Page 9: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/9.jpg)
Where we will go
• Forward genetics:– Genetic logic, complementation tests, nulls, going from mutant to gene– Dosage analysis. Dominance, structure function
analysis of domains (Greenwald lin-12 paper, a receptor, C. elegans)
– Enhancer/suppressor screens (Simon paper, Drosophila)
![Page 10: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/10.jpg)
Where we will go
• Reverse genetics:– SHP paper, AGL transcription factors, Arabidopsis
– Functional genomics
![Page 11: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/11.jpg)
Where we will go
• Genetic interactons:– Synthetic interactions (Lambie and Kimble, lin12-
glp-1)
– Allele-specific interactions
– Dose-specific interactions (Jorgensen)
– Point mutants versus nulls
– Epistasis-two class days
![Page 12: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/12.jpg)
How this part of the course will work
• Primary literature papers with homework questions
• Class discussion-be prepared to discuss any figure
• Group presentations
• Guest speakers
![Page 13: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/13.jpg)
How the course will end
• Research proposal:– Abstract and AIMS due before spring break (March 10)
• Identify a biological question
• AIM 1 must be a genome-wide mutagenesis
• AIMS 2/3 how you will test/analyze your mutants
– Rough draft due April
– Presentation
– Final paper
![Page 14: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/14.jpg)
The first step is to make an inbred strain. Why?
![Page 15: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/15.jpg)
The first step is to make an inbred strain. Why?
To make sure all of the parts are EXACTLY the same
![Page 16: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/16.jpg)
The second step is to find mutants
• What is the spontaneous rate of mutations per gene?
![Page 17: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/17.jpg)
The second step is to find mutants
• What is the spontaneous rate of mutations per gene?
• Looking at a single gene, 11/1,000,000 gametes have a mutation
• We use mutagens to increase that 1000 fold.
![Page 18: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/18.jpg)
Variation in strains is useful
• Natural variation can be used as a source of allelic variation
• Used commonly in agriculture and medicine
![Page 19: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/19.jpg)
Common mutagens
![Page 20: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/20.jpg)
Common mutagens
• EMS/MMS/NSG
• Transposons/T-DNA
• Ionizing radiation
• UV
• Spontaneous mutations
• DEB/Psoralen/ENU
![Page 21: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/21.jpg)
Common mutagens
• EMS/MMS/NSG
• Transposons/T-DNA
• Ionizing radiation
• UV
• Spontaneous mutations
• DEB/Psoralen/ENU
How do these affect DNA?
![Page 22: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/22.jpg)
Common mutagens
• EMS/MMS/NSG G to A transitions
• Transposons/T-DNA insert into gene
• Ionizing radiation breaks in DNA
• UV thymidine dimers
• Spontaneous mutations
• DEB/Psoralen/ENU gene-sized deletions of DNA
![Page 23: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/23.jpg)
Does every mutation result in a change in amino acid sequence?
![Page 24: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/24.jpg)
Does every mutation result in a change in amino acid sequence?
• No– Synonomous changes
• 3rd base wobble in codons• Some amino acids are specified by 6 triplets
![Page 25: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/25.jpg)
Does every change in an amino acid kill the protein?
Serine: UCXThreonine-ACX
![Page 26: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/26.jpg)
Does every change in an amino acid kill the protein?
• No, single base pair changes often lead to a change in a similar amino acid
![Page 27: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/27.jpg)
What kinds of mutations do you want?
![Page 28: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/28.jpg)
What kinds of mutations do you want?
• Nulls
• A variety of missense changes that might tell you about the roles of domains within that protein
![Page 29: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/29.jpg)
Nomenclature A
• Nonesense
• Missense
• Frameshift
• Knockout
• Null– Which inactivate proteins?– Which do you want?
![Page 30: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/30.jpg)
Nomenclature B
• Amorph
• Hypomorph
• Hypermorph
• Neomorph
![Page 31: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/31.jpg)
EMS-mechanism
![Page 32: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/32.jpg)
EMS-result
Most of time, any G can be changed to an A in either strand
![Page 33: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/33.jpg)
Which G-A changes can produce stop codons?
![Page 34: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/34.jpg)
Tryptophan: the cyanide capsule within many proteins
Glutamic Acid
![Page 35: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/35.jpg)
![Page 36: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/36.jpg)
What makes a good screen?
![Page 37: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/37.jpg)
What makes a good screen?
• Ease
• Precision-not too broad or too narrow
• Phenotypic followup
• Luck!
![Page 38: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/38.jpg)
![Page 39: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/39.jpg)
The Hartwell screen-perfect from the outset,or refined?
![Page 40: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/40.jpg)
Developmental screen logic
• Defects in an organ, in appearance
• Cell fate defects
• Mosaic versus signaling
![Page 41: APOG Today's schedule: SPIN How genetic dissection works](https://reader034.vdocuments.us/reader034/viewer/2022042814/5555e121d8b42a711f8b52b7/html5/thumbnails/41.jpg)
C. elegans websites
• http://www.wormatlas.org/userguides.html/lineage.htm
• http://www.wormclassroom.org/db/completeLineage.html
• http://www.wormclassroom.org/ac/transparent.html
• http://www.wormclassroom.org/intro.html