APOG

1
APOG
2
Todays 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

Genetics

• 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.)

4
Franss 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 ident
ify genes and understand their roles Precise mu
tate 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 I
ntrinsic logic-examine the roles of genes and
how they relate to one another, self-referenti
al
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
(No Transcript)
36
What makes a good screen?
37
What makes a good screen?

• Ease
• Precision-not too broad or too narrow
• Phenotypic followup
• Luck!

38
(No Transcript)
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.h
  tm
• http//www.wormclassroom.org/db/completeLineage.ht
  ml
• http//www.wormclassroom.org/ac/transparent.html
• http//www.wormclassroom.org/intro.html