APOG

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

• 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

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.

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.

Goals of genetic analysis

Identify components

Assign roles

Establish hierarchy

Build on the hierarchy

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.

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

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)

Where we will go

• Reverse genetics:– SHP paper, AGL transcription factors, Arabidopsis

– Functional genomics

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

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

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

The first step is to make an inbred strain. Why?

The first step is to make an inbred strain. Why?

To make sure all of the parts are EXACTLY the same

The second step is to find mutants

• What is the spontaneous rate of mutations per gene?

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.

Variation in strains is useful

• Natural variation can be used as a source of allelic variation

• Used commonly in agriculture and medicine

Common mutagens

Common mutagens

• EMS/MMS/NSG

• Transposons/T-DNA

• Ionizing radiation

• UV

• Spontaneous mutations

• DEB/Psoralen/ENU

Common mutagens

• EMS/MMS/NSG

• Transposons/T-DNA

• Ionizing radiation

• UV

• Spontaneous mutations

• DEB/Psoralen/ENU

How do these affect DNA?

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

Does every mutation result in a change in amino acid sequence?

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

Does every change in an amino acid kill the protein?

Serine: UCXThreonine-ACX

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

What kinds of mutations do you want?

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

Nomenclature A

• Nonesense

• Missense

• Frameshift

• Knockout

• Null– Which inactivate proteins?– Which do you want?

Nomenclature B

• Amorph

• Hypomorph

• Hypermorph

• Neomorph

EMS-mechanism

EMS-result

Most of time, any G can be changed to an A in either strand

Which G-A changes can produce stop codons?

Tryptophan: the cyanide capsule within many proteins

Glutamic Acid

What makes a good screen?

What makes a good screen?

• Ease

• Precision-not too broad or too narrow

• Phenotypic followup

• Luck!

The Hartwell screen-perfect from the outset,or refined?

Developmental screen logic

• Defects in an organ, in appearance

• Cell fate defects

• Mosaic versus signaling

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