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APOG Today's schedule: SPIN How genetic dissection works
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APOG Today's schedule: SPIN How genetic dissection works

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  • 1. APOG
  • 2. 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
  • 3.
    • 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
  • 4. 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.
  • 5. 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.
  • 6. Goals of genetic analysis
    • Identify components
    • Assign roles
    • Establish hierarchy
    • Build on the hierarchy
  • 7. 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.
  • 8. Logic and Rationale Comprehensive: all components Systematic: identify genes and understand their roles Precise: mutate one component at a time Powerful: remove one and only one component (and observe the consequences for function Certain: if approach is systematic and biology permits Valid: Intrinsic logic-examine the roles of genes and how they relate to one another, self-referential
  • 9. 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 )
  • 10. Where we will go
    • Reverse genetics:
      • SHP paper, AGL transcription factors, Arabidopsis
      • Functional genomics
  • 11. 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
  • 12. 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
  • 13. 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
  • 14. The first step is to make an inbred strain. Why?
  • 15. The first step is to make an inbred strain. Why? To make sure all of the parts are EXACTLY the same
  • 16. The second step is to find mutants
    • What is the spontaneous rate of mutations per gene?
  • 17. 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.
  • 18. Variation in strains is useful
    • Natural variation can be used as a source of allelic variation
    • Used commonly in agriculture and medicine
  • 19. Common mutagens
  • 20. Common mutagens
    • EMS/MMS/NSG
    • Transposons/T-DNA
    • Ionizing radiation
    • UV
    • Spontaneous mutations
    • DEB/Psoralen/ENU
  • 21. Common mutagens
    • EMS/MMS/NSG
    • Transposons/T-DNA
    • Ionizing radiation
    • UV
    • Spontaneous mutations
    • DEB/Psoralen/ENU
    How do these affect DNA?
  • 22. 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
  • 23. Does every mutation result in a change in amino acid sequence?
  • 24. 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
  • 25. Does every change in an amino acid kill the protein? Serine: UCX Threonine-ACX
  • 26. 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
  • 27. What kinds of mutations do you want?
  • 28. 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
  • 29. Nomenclature A
    • Nonesense
    • Missense
    • Frameshift
    • Knockout
    • Null
      • Which inactivate proteins?
      • Which do you want?
  • 30. Nomenclature B
    • Amorph
    • Hypomorph
    • Hypermorph
    • Neomorph
  • 31. EMS-mechanism
  • 32. EMS-result Most of time, any G can be changed to an A in either strand
  • 33. Which G-A changes can produce stop codons?
  • 34. Tryptophan: the cyanide capsule within many proteins Glutamic Acid
  • 35.  
  • 36. What makes a good screen?
  • 37. What makes a good screen?
    • Ease
    • Precision-not too broad or too narrow
    • Phenotypic followup
    • Luck!
  • 38.  
  • 39. The Hartwell screen- perfect from the outset, or refined?
  • 40. Developmental screen logic
    • Defects in an organ, in appearance
    • Cell fate defects
    • Mosaic versus signaling
  • 41. 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

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