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LECTURE 23: DISSECTION OF GENE FUNCTION I: GENETIC FINE STRUCTURE

LECTURE 23: DISSECTION OF GENE FUNCTION I: GENETIC FINE STRUCTURE. chapter 16 bead theory phage cross gene = unit of structure? gene = unit of change? gene = unit of function? summary problems. BEAD THEORY. bead theory : chromosome = linear (1-D) array of genes

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LECTURE 23: DISSECTION OF GENE FUNCTION I: GENETIC FINE STRUCTURE

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  1. LECTURE 23: DISSECTION OF GENE FUNCTION I: GENETIC FINE STRUCTURE • chapter 16 • bead theory • phage cross • gene = unit of structure? • gene = unit of change? • gene = unit of function? • summary • problems

  2. BEAD THEORY • bead theory: chromosome = linear (1-D) array of genes • genes are defined by mutant alleles that ... • affect single characters / traits • map to single locus on chromosome • result in mutant phenotypes when individuals are homozygous recessive • show Mendelian ratios in progeny of crosses

  3. BEAD THEORY • testable bead theory hypotheses: 1. gene as fundamental unit of structure... indivisible by crossing over 2. gene as fundamental unit of change... mutations change alleles from one form to another; there are no smaller units within genes that can change 3. gene as fundamental unit of function... parts of genes cannot function alone in tests of complementation

  4. PHAGE CROSS • Benzer (50’s) studied rII gene of T4 bacteriophage infecting E. coli hosts to test the bead theory... • began as physicist (like Delbrück, Brenner & others) • looking for physical properties unique to living matter • most important & influential geneticist (personal bias) • rigorous reductionist approach from physics • made most important link between classical / Mendelian genetics & modern molecular biology • now looks for genes to extend lifespan (in flies)

  5. PHAGE CROSS • Benzer (50’s) studied rII gene of T4 bacteriophage infecting E. coli hosts to test these hypotheses...

  6. PHAGE CROSS • Benzer (50’s) studied rII gene of T4 bacteriophage infecting E. coli hosts to test these hypotheses... • rII mutants are conditional... grow in E. coli B • B is permissive host

  7. PHAGE CROSS • Benzer (50’s) studied rII gene of T4 bacteriophage infecting E. coli hosts to test these hypotheses... • rII mutants are conditional... grow in E. coli B but not K • B is permissive host, K is restrictive host

  8. PHAGE CROSS • Benzer (50’s) studied rII gene of T4 bacteriophage infecting E. coli hosts to test these hypotheses... large, round small, ragged

  9. GENE = UNIT OF STRUCTURE? • hypothesis #1: gene is fundamental unit of structure... indivisible by crossing over ?

  10. GENE = UNIT OF STRUCTURE? • hypothesis #1: gene is fundamental unit of change... indivisible by crossing over ? • PFU on B = total, PFU on K = ½ of recombinants • recomb. frequency (RF) = 2(PFU on K) /PFU on B

  11. GENE = UNIT OF STRUCTURE? • hypothesis #1: gene is fundamental unit of structure... indivisible by crossing over ? • PFU on B = total, PFU on K = ½ of recombinants • recomb. frequency (RF) = 2(PFU on K) /PFU on B

  12. GENE = UNIT OF STRUCTURE? • what is the smallest unit of recombination detectable? • phage system can detect 1 mutation in 109 progeny • recombination frequency (RF) = 1 x 10–9 •  smallest distance = 1 x 10–7 cM • if 1 cM  5 x 105 bp (on average, in flies) •  bp are separated by 2 x 10–6 cM • phage system ~20x more powerful than needed to detect smallest possible distance (which we now know is adjacent base pairs) • phage is the ultimate genetic system ... resolution!!

  13. GENE = UNIT OF STRUCTURE? • hypothesis #1: gene is fundamental unit of structure... indivisible by crossing over ? ... 

  14. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? • same experimental protocol as for #1

  15. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? • same experimental protocol as for #1

  16. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? • same experimental protocol as for #1 • mutants from #1 were deletions when they did not ... • recombine with some other mutants • ever revert back to wild type • deletions used to map positions of mutational sites

  17. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? all survive on... B only

  18. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? 4 + + 5

  19. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? 4 + + 5

  20. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? 4 5 survive on... B only K & B + +

  21. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? 4 +

  22. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? 4 + survive on... B only K & B

  23. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? 4 +

  24. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? 4 +

  25. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? no recombination 4 + survive on... B only B only

  26. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 D1 D2 D3 D4

  27. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 D1 D2 D3 D4 A B C D E F G

  28. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ?

  29. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene ? ... 

  30. GENE = UNIT OF CHANGE? • hypothesis #2: gene is fundamental unit of change... mutations change alleles from one form to another; there are no smaller mutable units within a gene...  • point mutations (= revert to wild type at low frequency) mapped to regions defined by deletion mutations • point mutations in the same regions that did not recombine with each other were identical sites • distribution of rII point mutations was not random...

  31. GENE = UNIT OF CHANGE? • distribution of rII point mutations was not random... • hot spots & cold spots

  32. GENE = UNIT OF CHANGE? • Poisson distribution predicted missed cold spots • observed hot spots (e.g.) • site = nucleotide base pair

  33. GENE = UNIT OF CHANGE? • summary of fine structure mapping of rII region in T4

  34. GENE = UNIT OF FUNCTION? • hypothesis #3: gene is fundamental unit of function... parts of genes cannot function in complementation tests ?

  35. GENE = UNIT OF FUNCTION? • hypothesis #3: gene is fundamental unit of function... parts of genes cannot function in complementation tests ?... • mutants fell into two functional groups... 2 genes • mutations fail to complement others in the same group 

  36. SUMMARY • complementation or recombination ? • complementation... • protein level, mixing of gene products • no change in genotypes relative to parents • requires 1 generation of crosses to investigate • recombination... • DNA level • results in new genotypes relative to parents • requires 2 generations of crosses to investigate

  37. SUMMARY • complementation or recombination? • test single phage from E. coli K lysate (F1) for infection on E. coli B & E. coli K (F2)

  38. SUMMARY • if you are dealing with complementation (only)... orhigh F1 count no F1 all F1 F2 plaques on E. coli B but all still mutant, no F1 F2 plaques on E. coli K

  39. SUMMARY • if you are dealing with recombination (only)... low F1 count but all recombinant F1 F2 plaques on E. coli B

  40. SUMMARY • if you are dealing with recombination (only)... low F1 count only ½ recombinant F1 F2 plaques on E. coli K

  41. SUMMARY • reality... recombination & complementation ... orhigh F1 count low F1 all F1 F2 plaques on E. coli B

  42. SUMMARY • reality... recombination & complementation ... orhigh F1 count low F1 all F1 F2 plaques on E. coli B only ½ recombinant F1 F2 plaques on E. coli K

  43. SUMMARY • testable bead theory hypotheses: 1. gene as fundamental unit of structure... indivisible by crossing over 2. gene as fundamental unit of change... mutations change alleles from one form to another; there are no smaller units within genes that can change 3. gene as fundamental unit of function... parts of genes cannot function alone in tests of complementation   

  44. PROBLEMS • stay tuned...

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