lecture 23: dissection of gene function i: genetic fine structure chapter 16 bead theory phage...

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

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

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

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)

PHAGE CROSS

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

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

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

PHAGE CROSS

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

large, roundsmall, ragged

PHAGE CROSS

hypothesis #1: gene is fundamental unit of structure... indivisible by crossing over ?

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

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

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!!

GENE = UNIT OF STRUCTURE?

hypothesis #1: gene is fundamental unit of structure... indivisible by crossing over ? ...

GENE = UNIT OF STRUCTURE?

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

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

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

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 all survive on...B only

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

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

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 onlyK & B

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 +

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 onlyK & B

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 +

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 +

GENE = UNIT OF CHANGE?

4 +

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 onlyB only

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

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

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 ?

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 ? ...

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...

GENE = UNIT OF CHANGE?

distribution of rII point mutations was not random... hot spots & cold spots

GENE = UNIT OF CHANGE?

Poisson distribution predicted missed cold spots observed hot

spots (e.g.)

GENE = UNIT OF CHANGE?

site = nucleotide base pair

summary of fine structure mapping of rII region in T4

GENE = UNIT OF CHANGE?

hypothesis #3: gene is fundamental unit of function... parts of genes cannot function in complementation tests ?

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

GENE = UNIT OF FUNCTION?

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

SUMMARY

complementation or recombination?

test single phage from E. coli K lysate (F1) for infection on E. coli B & E. coli K (F2)

SUMMARY

if you are dealing with complementation (only)...

SUMMARY

or high F1 countno F1

all F1 F2 plaques on E. coli Bbut all still mutant, no F1 F2 plaques on E. coli K

if you are dealing with recombination (only)...

SUMMARY

low F1 count

but all recombinant F1 F2 plaques on E. coli B

if you are dealing with recombination (only)...

SUMMARY

low F1 count

only ½ recombinant F1 F2 plaques on E. coli K

reality... recombination & complementation ...

SUMMARY

or high F1 countlow F1

all F1 F2 plaques on E. coli B

reality... recombination & complementation ...

SUMMARY

or high F1 countlow F1

all F1 F2 plaques on E. coli Bonly ½ recombinant F1 F2 plaques on E. coli K

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

PROBLEMS

stay tuned...