The Fine Structure of a Gene by Seymour Benzer Scientific American, January 1962 pg. 2 - 15

1. The Fine Structure of a Geneby Seymour BenzerScientific American, January 1962 pg. 2 - 15 Genet 270 September 20, 2012

2. Outline • Background the T4 phage the rII mutation • Procedures • deletion mapping • cis-trans complementation • mutation rates • mutagenesis Summary

3. Some Important Terms • Temperate virus • infection of host does not necessarily cause lysis but whose genome may replicate together with that of the host • Prophage • genome of a temperate virus when it is replicating together with that of the host (usually integrated) • Lysogen • a bacteria containing a prophage

4. T4 replication process Attachment of virus to host cell Injection of viral DNA

5. T4 replication process Lysis of host cell Synthesis and assembly of new viruses using host machinery Replication of viral DNA Production of early enzymes

6. The T4 Phage

7. The T4 Phage

8. Benzer’s work • Fine structure mapping of the T4 genome, focusing on the rII region, by: • dividing it into functional units • determining the mutation rates of the region

9. r- The rII Mutant • rII mutants are easily identified by the appearance of the plaques they form on a bacterial culture r+

10. The rII Mutant • a class of T4 phage mutants • a conditional lethal mutation

11. cis-trans complementation

12. CIS (same DNA molecule) active active active TRANS (different DNA molecules) inactive active active cis-trans complementation DIFFERENT functional unit DIFFERENT functional unit SAME functional unit

13. Cistron A Cistron B Mutation and recombination sites rII region Results of cis-trans comparison of the rII region

14. Practice Question 1 Mutant 1 x 2 = plaques Mutant 1 x 3 = plaques Mutant 2 x 3 = no plaques Given the above results from the trans portion of a cis-trans test, where can you draw the cistron boundary? 2 3

15. Point vs. Deletion Mutations • Point mutations • mutations involving 1 bp • Reversible • Deletion mutations • loss of large segments of DNA • not reversible

16. Deletion Mapping Co-infect E. coli strain B (permissive host) with two different mutant phage: one containing a point mutation, the other a deletion to allow for recombination Plate recombinants on strain K (restrictive host) NO OVERLAP OVERLAP 2 1 X Plate on strain K

17. Deletion Mapping • By mapping point mutations against progressively narrower deletions, Benzer was able to determine their location in the genome in less crosses. Large deletions Progressively smaller deletions Point mutations within that narrow region

18. Practice Question #1 • Which deletion(s) would be most informative for identifying the location of point mutation X?

20. E.coli B E. coli K - +/- + Strain B (permissive) + - - -/+ + X - + -/- - + + + +/+ Total progeny Wild-type progeny 2 x Wild-type Recombination Frequency = Total Intragenic mapping • Recombination frequency # Recombinants Recombination Frequency = Total  distance =  recombination =  wt

21. Are all sites equally susceptible to mutation? • Map many spontaneous rII mutants (deletion mapping) • Mutations that cannot undergo recombination affect the same site • Counted how many independent repeats occurred at different sites. Sites Mutations Equal Random Unequal Hot Spots

22. Mutation sites “hot spots” for mutation

23. Effect of mutagens on rII sites • Method: • Expose rII mutants to a mutagen • How many revert to wild-type? • Result: • mutagens are specific • Example: • 5-bromouracil • substitutes for T

24. Summary • Deletion mapping allows fine dissection of the genome • cis-trans test can determine if two mutations are in the same functional unit • a cistron is a hereditary unit that functions independently • some areas of the genome are more susceptible to mutation than others

25. Questions?? • Next week: Quiz! Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA