What’s in store? Mutation: Where do they come from? Mutation: How do we get rid of them? Cancer: The result of mutation Thanksgiving Genetic variation Genetics of populations 2 nd UR Symposium on Agrobacterial genetics Exam IV

What’s in store today? Is mutatagenesis random or induced? Luria-Delbrück fluctuation experiment Lederbergs’ replica plating experiment What mutations arise spontaneously? What is the cause of spontaneous base substitutions? Spectrum of mutations in lacI Tautomerization Transitions vs transversions

Nature of mutation Luria-Delbrück fluctuation experiment Culture growing without lactose Culture able to metabolize lactose + lactose Person sensitive to smallpox Acquired immunity + virus Culture sensitive to bacteriophage + phage Acquired hereditary immunity + phage Culture sensitive to bacteriophage sensitive cells rare mutants Clonal growth of random mutants vs.

Nature of mutation Luria-Delbrück fluctuation experiment Culture sensitive to bacteriophage + phage Acquired hereditary immunity + phage Culture sensitive to bacteriophage sensitive cells rare mutants Clonal growth of random mutants vs. How to distinguish the two?

Nature of mutation Luria-Delbrück fluctuation experiment Little variation from tube to tube Great variation from tube to tube Add phage

SQ2: What if tubes pooled? Add phage Tube 1 Tube 2 Tube 3 Tube 4 4 mutants 5 mutants 5 mutants 6 mutants

Nature of mutation Lederbergs’ replica plating experiment Acquired hereditary immunityRandom mutation

How to get lacI mutants Single cell Single colony Culture PGal + Colonies Isolate DNA Sequence lacI SQ6: Why a single colony?

SQ8: What kind of mutations do you expect? What kind of mutations did Crick et al see? Problem set 4: Mutation of Factor VIII gene Wild-type5'-GGAGTTGAGTCATGGACTCTAAGCAGCGATCCACAAAG... Individual a 5'-GGAGTT T AGTCATGGACTCTAAGCAGCGATCCACAAAG... Individual b 5'-GGAGTTGAGTCAT T GACTCTAAGCAGCGATCCACAAAG... Individual c 5'-GGAGTTGAGTCATGGACTCT T AGCAGCGATCCACAAAG... Individual d 5'-GGAGTTGAGTCATGGACTCTAAGCAGC T ATCCACAAAG... Individual e 5'-GGAGTTGAGTCATGGACTCTAAGCAGCGATCCAC T AAG... What kind of mutation has been cropping up all semester? Base substitution rIIA - rIIA + proflavin Single base insertion/deletion What kind of mutations were in your T4 tester strains? Big deletions What kind of mutations were actually observed in lacI?

GTCTGGCTGGCTGGCTGGC GTCTGGCTGGCTGGC Wild type Duplication SQ9 See anything unusual?

GTCTGGCTGGC GTCTGGCTGGCTGGC Wild type Deletion

SQ6: Why start with a single colony? Single cell Single colony

SQ6: Why start with a single colony? Consider the alternative Concentration?2·10 9 cells/ml Number of cells transferred?> 2·10 6 cells Number of mutants transferred?> 4 mutants Number of mutants after regrowth of culture?> 4000 mutants

Tautomerization of bases C T G A C* T* A G

Mutagenesis from tautomeric base

Effects of transition mutations

Effects of transversion mutations