Lecture F - Mutations

Genetics - second lecture

DNA repair mechanisms

reduce mutation rate

Base Excision Repair

DNA Glycosylase removes base (recognizes U)

1. C deaminated to U recognized

2. Glycosylase removed U, leaves AP site

3. AP endonuclease makes nick

4. DHA exonucleases creates gap

5. DNA poly fills gap

6. DNA ligase seals gap

Nucleotide Excision Repair

1. UvrA and UvrB proteins scan for distortions 

2. UvrB and UvrC remove the distorted (Thymine dimers) strand

3. the good strand is used for template repair 

UvrB and C remove 12 nucleotides spanning mutation

DNA repair mechanisms need DNA to be 

double stranded 

• the good strand is used to insert bases after replication 

Mismatch repair (MMR)

Excises and replaces replication mistakes

1. parent strands marked with methyl groups

2. MutS and MutL recognize a mismatch in replicated DNA

3. MutL recruits MutH, who makes a nick in strand opposite to the methyl tag

4. DNA exonucleases excise DNA from unmethylated new strand

5. repair and methylation of newly synthesized DNA strand

MMR Problem + solution

During repair, they know one base is wrong but don’t know which one is wrong and right

• the distortion can be in the original or new DNA

MMR - Bacteria parent vs new

Parental/Original - Methylated

New - unmethylated

• the unmethylated is likely the one to be repaired

After proofreading by polymerase and MMR, one mistake occurs every

10⁹ bases

Why bacteria has 2 life cycles

If there are many bacteria or few resources, it is not worth generating bacteria.

• bacteria will stay in lysogenic cycle

Lytic Cycle

1. bacteriophage injects phage DNA 

2. circularizes and makes copies of itself 

3. goes through replication/transcription/translation

4. lysis

Lysogenic Cycle

1. bacteriophage injects phage DNA

2. circularizes and makes copies of itself

3. forms a prophage

4. can excise depending on resource availability

Phage growth on bacterial lawn

Consists of many bacterial cells

• phage capsid injects phage DNA into a cell

• each infected cell lyses and releases a phage that can infect nearby cells

• Nearby cells lyse, infecting more cells (shown as plaque)

Prophage

genetic material of bacteriophage integrated in the host bacterium

Plaque

where the bacteria have been lysed

• shows up clear

Plaque Morphology: RII+

Wild-type

Small plaques

Plaque Morphology: RII-

Mutation

Large Plaques —> Rapid Lysis

• has less phage in them

Plaque Morphology: h+

Wild-type 

• turbid

Plaque Morphology: h-

mutation

• clear

rII infection of E.coli K12 (λ)

Wildtype can infect E.coli K12 (λ)

rII mutant cannot infect E.coli K12 (λ)

Complementation test in phages

tests if 2 mutants of the same phenotype have the same gene or not

• are all rII mutants in the same gene?

How do phages cross with each other?

coinfection

Difficult of phage complementation

There is only a single copy of the genes

coinfection

How phages cross

• the genomes of two mutants in the same cell

No plaque growth

• No complementation occurred

• mutations in same gene

Plaque growth

• complementation occurred

• mutations in different genes

coinfection leading to no growth

coinfection between mutant 1 and mutant 2 (mutations on same gene (B)) 

• you get 2 mutated B and wt A

cannot grow in E.coli K12 (λ)

No plaque —> mutations on same gene

Coinfection leading to growth

Coinfection between mutant 1 and mutant 3 (mutations on different genes (B and A)

• you get one double mutant and one wt copy

• coinfecttion on E.coli K12 (λ) results in plaque growth

plaque growth —> mutations on different genes

Intergenic Mapping

Distance between different genes

• measured by the recombination between specific alleles of different genes

Number of recombinants/total progeny = recombinant frequency

Recombinant frequncy x 100 = distance

Mapping is more difficult in bacteriophages

Need two chromosomes with two different alleles, we only have one chromosome so coinfection with two different phage strains is required

Intragenic Mapping

Recombination between different mutations in the SAME gene

• 2 mutations in the same gene and have the same phenotype

• uses recombination frequency to determine how close the mutations are in the gene

Intragenic Mapping: Recombination

The closer the mutations, the rarer the crossing over

Intragenic Mapping: wild-type is produced

Recombination occurred

• double mutant and wild-type are present

Seymour Benzer and the rII genes of T4

Generated thousands of mutants and mapped them to see where in the same gene they are located for the rIIA and rIIB genes 

Intragenic Recombination and Mapping at rIIA locus

Draw diagram/understand it

Complementation vs intragenic recombination

Complementation: Immediately coinfects and grows strands on E.coli K12 (λ) to see if genomes produce genes 

Intragenic: Between two mutants of the same gene, grown on B strain to recombine and then coinfects

Mapping mutants in rII gene

1. coinfect strain B with two rII mutants of same gene - infect strain B and grow it in the culture

2. Collect the new phage - will have recombinants, and progeny

3. Plate on strain B and E.coli K12 (λ)

Mapping mutants in rII gene: Contents of phage collections

Nonrecombinants mostly - Parentals

recombinants - wild-type and double mutant phages will be identical in amount

Mapping mutants in the rII gene: Recombination Frequency

Recombination Frequency = # of plaques on E.coli K12 (λ) / # of plaques on strain B  X 2

Why multiply by 2 in recombination frequency

to account or the double mutants as they go undetected

Mapping mutants in the rII gene: map distance between mutants

multiply recombination frequency by 100

Deletions helped to group point mutants to regions

Mutagens can form small deletions and point mutations

• if point mutation is within deleted region no wt will reappear

• if point mutation is outside deleted region wt will appear

Crossing two point mutations

wt can appear

• wt and double mutant can occur

Crossing point mutation and deletion

Nothing to recombine with

• deletion takes out the region with both mutations 

• will not revert back to wt

Results of intragenic mapping of rII

• recombination can probably take place between any pair of adjacent nucleotides

• genes are a linear array of nucleotides

One hot spot equal how many mutations

500 independent mutations 

Results of intragenic mapping of rII:

why are some locations of same phenotype hot spots, and others find no mutations

• some locations are not mutants since the EMS causes C to T changes so areas without mutants may be A/T

• some may not be important in the function

Who established the link between genes and polypeptides? 

Garrod

Garrod’s analysis of a human disease suggested

genes code for enzymes

• link between genes and polypeptides

Alkaptonuria - wt and mutations

Genetic disease

• recessive alleles in individuals who are homozygous for the allele cannot produce the enzyme

• the wildtype is required to produce homogenetic acid 

Alkaptonuria - pathway

1. occurs as urine that blackens

2. this happens due to the absence of the homogenestic acid oxidase

3. homogenestic acid produces maleylacetoacetic acid

Beadle and Tatum

Proposed one gene one enzyme hypothesis

Does a single gene control pathways or do four genes, encoding one of the enzymes?

single gene controlled the synthesis of a single enzyme

Beadle and Tatum general experiment goal

Generated mutants in neurospora to investigate the genetics of metabolic pathways 

Beadle and Tatum Set Up

Experimental organism: Neurospora crassa (bread mold)

Inherited traits: nutritional requirements

• wild type requires:

  • carbon source (sugar)

  • inorganic salts

  • biotin (vitamin)

Beadle and Tatum Hypothesis

1. neurospora has pathways with enzymes to convert simple molecules into those required for life

2. has genes controlling synthesis/activity of these enzymes

Beadle and Tatum Experiment

1. Investigate known metabolic pathways

2. generate mutants defective in ability to synthesize the final product of the pathway

3. Complementation tests: determine if mutants are in different genes

4. Determine if each mutation disrupts all the steps in the pathway or just one

Beadle and Tatum - What they did in their experiment

Generated mutants unable to synthesize arginine

1. mutagenize (exposed to xray)

2. isolate individuals on complete medium

3. identify nutritional mutants: test for inability to grow on minimal medium

4. Identify nutritional requirements of the mutant: grow on minimal medium plus one amino acid

5. since arginine grew a spore, we know that there are mutants unable to synthesize arginine 

Different mutants affecting different steps rather than whole pathway

1. substances produced after the genetic block will enable growth and some mutants are rescued by intermediates but not others

2. The earlier the mutated gene acts in a metabolic pathway, the more the intermediary substances will be able to use for growth

3. Mutants carrying mutations in genes later in the pathway are able to use fewer substances that are produced at the end of the pathway

4. substances produced towards the end of the pathway can be utilized by more different mutants (and vise versa)

Neel and Beet

Determined sickle cell anemia is a genetic disease

Ingram

Disease is associated with a change in the sixth amino acid of the hemoglobin B-chain (glutamic acid to valine)

HbA/HbA

Normal

HbA/HbS

sickle cell trait

HbS/HbS

Sickle cell anemic

normal vs sickle cell individual: N-terminus

Normal: Val-His-Leu-Pro-Glu-Glu

Sickle-cell Individual: Val-His-Leu-Pro-Val-Glu

normal vs sickle cell individual: Protein structure

Glu to Val (changes) 

normal vs sickle cell individual: Aggregation and solubility

Sickle-cell individual: proteins aggregate to long fibers

Sickle-cell individuals: less soluble hemoglobin

normal vs sickle cell individual: Cell shape

Normal: round, normal RBC w divot in it

Sickle cell: long, fiber (poop) shaped