APOG

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APOG
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Homologous recombinationand plants

• There is no gene replacement as in budding yeast
  or mammals.

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Plants and light
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What makes a good screen?
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What makes a good screen?

• A phenotype with interesting biology
• Ease
• Precision-not too broad or too narrow
• Phenotypic followup
• Luck!

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The Hartwell screen- perfect from the outset, or
refined?
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Wildtype
cav1-2
cav1-5
0-20weak blue _at_60 strong blue light
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The first step is to make an inbred strain. Why?
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The first step is to make an inbred strain. Why?
To make sure all of the parts are EXACTLY the
same
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The second step is to find mutants

• What is the spontaneous rate of mutations per
  gene?

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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 100 to 1000 fold.

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Common mutagens
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Common mutagens

• EMS/MMS/NSG
• Transposons/T-DNA
• Ionizing radiation
• UV
• Spontaneous mutations
• DEB/Psoralen/ENU

How do these affect DNA/genes?
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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

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EMS-mechanism
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EMS-result
Most of time, any G can be changed to an A in
either strand
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Does every mutation result in a change in amino
acid sequence?
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Does every mutation result in a change in amino
acid sequence?

• No
• Synonomous changes UCX-Thr
• 3rd base wobble in codons

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Does every change in an amino acid kill the
protein?
Serine UCX Threonine-ACX
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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

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What kinds of mutations do you want?
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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

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What kinds of mutations produce nulls?
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What kinds of mutations produce nulls?

• Early stop codons
• Frameshifts
• Splice site mutations
• Missense changes in key residues
• insertions

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What is the best molecular test for a null?
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What is the best molecular test for a null?

• No protein
• No mRNA
• Part of coding region missing

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Nomenclature A

• Nonesense
• Missense
• Frameshift
• Knockout
• Null
• Which inactivate proteins
• How?
• Which do you want?

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Nomenclature B

• Amorph
• Hypomorph
• Hypermorph
• Neomorph

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Which G-A changes (from EMS/MMS can produce stop
codons?
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Tryptophan and Glutamine (CAG) the cyanide
capsules within many proteins
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How do you know the phenotype is due to only one
mutation?
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How do you know the phenotype is due to only one
mutation?

• Segregates as a single nuclear recessive

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Determining the number of genes picked up by your
screen
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Determining the number of genes picked up by your
screen

• Complementation test

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Determining the number of genes picked up by your
screen

• Complementation test
• Does it ever fail?

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Non-allelic non-complementation

• Alpha and Beta tubulin
• Interacting proteins at synapses
• Heterodimerizing transcription factors

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Avoiding failure
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Avoiding failure

• Sequencing mutations
• Mapping mutants

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Going from mutant to the gene in Arabidopsis

• Tagging
• Map-based cloning
• Confirmation by reverse genetics

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How do you determine the location of your gene
with closely linked markers and few recombinants?

• X 4/1000
• YFG
• Z 2/1000

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How do you know when you are getting all possible
components in your screen?
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How do you know when you are getting all possible
components in your screen?
Poisson distribution
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How did they prove CAV1 is NPL1?
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Proving CAV1 is NPL1

• Genetic complementation test
• Sequence mutations
• They could have performed a different kind of
  complementation test-rescue mutant with the
  wild-type gene.

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cav1 mutants still have weak blue light
accumulation Response This response is mediated
by NPH1 and NPL1.
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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