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LECTURE 23: DISSECTION OF GENE FUNCTION I: GENETIC FINE STRUCTURE  chapter 16  bead theory  phage cross  gene = unit of structure?  gene = unit of.

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Presentation on theme: "LECTURE 23: DISSECTION OF GENE FUNCTION I: GENETIC FINE STRUCTURE  chapter 16  bead theory  phage cross  gene = unit of structure?  gene = unit of."— Presentation transcript:

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

3 F 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 BEAD THEORY

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

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

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

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

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

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

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

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

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

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

14 F 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 ? F same experimental protocol as for #1 GENE = UNIT OF CHANGE?

15 F 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 ? F same experimental protocol as for #1 GENE = UNIT OF CHANGE?

16 F 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 ? F same experimental protocol as for #1 F mutants from #1 were deletions when they did not... g recombine with some other mutants g ever revert back to wild type F deletions used to map positions of mutational sites GENE = UNIT OF CHANGE?

17 F 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 GENE = UNIT OF CHANGE?

18 F 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 ? GENE = UNIT OF CHANGE?

19 F 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 ? GENE = UNIT OF CHANGE?

20 F 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 ? survive on... B only K & B GENE = UNIT OF CHANGE?

21 F 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 + GENE = UNIT OF CHANGE?

22 F 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 GENE = UNIT OF CHANGE?

23 F 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 + GENE = UNIT OF CHANGE?

24 F 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 + GENE = UNIT OF CHANGE?

25 4 + F 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 survive on... B only GENE = UNIT OF CHANGE?

26 F 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 ? D1 D2 D3 D4 GENE = UNIT OF CHANGE?

27 F 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 ? D1 D2 D3 D4 A B C D E F G GENE = UNIT OF CHANGE?

28 F 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 ? GENE = UNIT OF CHANGE?

29 F 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 ?...  GENE = UNIT OF CHANGE?

30 F 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...  F point mutations (= revert to wild type at low frequency) mapped to regions defined by deletion mutations F point mutations in the same regions that did not recombine with each other were identical sites F distribution of rII point mutations was not random... GENE = UNIT OF CHANGE?

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

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

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

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

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

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

37 F complementation or recombination? F test single phage from E. coli K lysate (F 1 ) for infection on E. coli B & E. coli K (F 2 ) SUMMARY

38 F if you are dealing with complementation (only)... SUMMARY or high F 1 count  no F 1 all F 1  F 2 plaques on E. coli B but all still mutant,  no F 1  F 2 plaques on E. coli K

39 F if you are dealing with recombination (only)... SUMMARY low F 1 count but all recombinant F 1  F 2 plaques on E. coli B

40 F if you are dealing with recombination (only)... SUMMARY low F 1 count only ½ recombinant F 1  F 2 plaques on E. coli K

41 F reality... recombination & complementation... SUMMARY or high F 1 count  low F 1 all F 1  F 2 plaques on E. coli B

42 F reality... recombination & complementation... SUMMARY or high F 1 count  low F 1 all F 1  F 2 plaques on E. coli B only ½ recombinant F 1  F 2 plaques on E. coli K

43 F 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 SUMMARY   

44 PROBLEMS  stay tuned...


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