genetics exam 3

jean baptiste lamarck

• physiological adaption

• proposed that physiological events determine whether traits are passed along to offspring

• long necked giraffes evolved by stretching their necks to reach higher trees

charles darwin

• random mutations

• genetic variation occurs by chance

• natural selection results in better-adapted organisms 

• long-necked giraffes are randomly born and have more offspring due to their competitive advantage

lamarck’s theory on evolution

• law of use and disuse: body parts got stronger or deteriorated depending on whether it was sued or not

• inheritance of acquired characteristics: acquired changes were passed onto offspring 

lederberg experiment 

• tonr - E.coli resistant to infection by bacteriophage T1

• physiological adaptation theory: predicts number of tonr is very low unless theres selection 

• random mutation theory: predicts number of tonr fluctuate in different bacterial population and will occur without selection 

• velvet is pressed onto master plate to collect bacterial cells 

• transfer bacteria to selective and non-selective plates  

• selection is resistant to phage T1

• resistant colonies are on both the non-selective and selective plates

• mutations didn’t occur bc the cell needed them, they were there before any contact with the selective medium  

mutations happen randomly 

• statistically random events 

• occurs with no relation to any adaptive advantage 

• potentially favorable mutation doesn’t arise because the organism has a need for it 

sickle cell anemia 

• single point mutation 

• causes hemoglobin molecules to crystallize when oxygen levels in the blood are low 

• makes red blood cells sickle 

mutations in coding sequences 

• missense mutation: point mutation changes amino acid 

• nonsense mutation: point mutation produces stop codon

• silent mutation: point mutation but doesn’t alter the genome

mutations outside coding sequences

• up mutations: increase expression

• down mutation: decrease expression

deleterious mutations

• decrease chances of survival

• most extreme are lethal mutation

beneficial mutations 

• enhance the survival or reproductive success of an organism 

neutral mutations

• has no effect on the chances of survival

conditional mutations

• affect the phenotype only under a defined set of conditions

• ex: temp-sensitive mutation: environment can affect whether a given mutation is deleterious or beneficial or neutral

forward mutation 

• changes the wild-type genotype into some new mutation 

reverse mutation

• changes a mutant allele back to the wild-type

• genotype is changed back

suppressor mutations

• can change back the phenotype but the genotype is not reversed

• a second mutation at a different location will sometimes counteract the effects of a first mutation → passing on both traits

• intragenic and intergenic

intragenic suppressors 

• the second mutant site is within the same gene as the first mutation 

• ex: transporter protein:

  • first mutation disrupts normal protein function

  • second suppressor mutation affecting the same protein restores function

intergenic suppressor

• second mutant site is in different gene from the first mutation

intergenic suppressor: redundant function 

• first mutation inhibits the function of a protein 

• second mutation alters a different protein to carry out that function 

intergenic suppressor: common pathway

• two or more different proteins may function as enzymes in a common pathway

• mutation that causes a defect in one enzyme may be compensated for by a mutation that increases the function of a different enzyme in the same pathway

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intergenic pathway: multimeric protein

• mutation in a gene encoding one protein subunit that inhibits function

• may be suppressed by a mutation in a gene that encodes a different subunit

• the double mutant has restored function

intergenic suppressor: transcription factor

• first mutation causes loss of function of a particular protein

• second mutation may alter a transcription factor and cause it to activate the expression of another gene 

• this other gene encodes a protein that can compensate for the loss of function caused by the first mutation 

germ-line mutations

• occurs before fertilization

• mutation is found throughout the entire body

• half the gametes carry the mutation

• therefore the mutation can be passed on to future generations

somatic mutations

• occurs after fertilization

• patch of affected area

• the size of the patch depends on the timing of the mutation (earlier → larger patch)

• genetic mosaic: somatic regions that are genotypically different from each other

• none of the gametes carry the mutation

• mutation cannot be passed on to future generations

transition mutations 

• purine changes to a different purine 

• pyrimidine changes to different pyrimidine 

• less detrimental at the 3rd codon position bc pyrimidine never changes a code and purine only changes 2 codes

transversion mutations

• purine and pyrimidine are interchanged

• more detrimental because they are more likely to change an amino acid 

spontaneous mutations 

• random, unpredictable events 

• natural changes/error 

• replication errors or chemical changes 

induced mutations

• caused by environmental agents

• chemical, radiation

• increased rate of mutation (probability of a particular type of mutation per unit time or gen)

depurination

• Apurimic or abasic (AP) site

• bond between the base and sugar is broken

• extremely common spontaneous mutation

• fixed by base excision repair

deamination 

• happens spontaneously but can be induced by nitrous acid 

• removal of an amino group 

• deaminated cytosine becomes uracil 

• deaminated methylcytosine becomes thymine 

  • causes CG to AT → not detected by repair mechanisms

  • involved in epigenetics

replication mutations 

• DNA polymerase occasionally inserts incorrect nucleotides due to mispairing 

• leads to point mutations 

• most get fixed by proofreading 

• slippage during replication can lead to small insertions or deletions 

• usually at repeats of the same nucleotide 

strand slippage during replication

• occurs at nucleotide repeats

• causes small insertions or deletions

one nucleotide loops out

• on new strand (slips out) - insertion

  • polymerase moves past the point of the loop and just keeps reading the next base on the template strand

  • new strand is misaligned so the polymerase ends up copying a template base twice

  • looped nucleotide becomes a permanent, un-paired insertion in the final DNA

• on old strand (skipped)- deletion

  • polymerase is reading the template strand, but it skips right over the looped-out base

  • polymerase doesn't "see" the looped base, so it doesn't add anything to the new strand to pair with it

  • looped-out base is permanently skipped and deleted from the genetic sequence in the new copy

both causes frameshift mutations

Trinucleotide Repeat Expansion

• due to replication slippage 

• fragile X syndrome (CGG) 

• The neurological disorders myotonic dystrophy (CTG)

• Kennedy disease (AGC)

• Friedreich ataxia (AAG)

• Spinocerebellar ataxia type 1 (AGC)

• Huntington disease (AGC)

fragile x syndrome

• Symptoms may include: learning disabilities or autistic-like behavior, emotional problems, and physical abnormalities such as prominent ears, a long, narrow face, flexible fingers

• Loss-of-function of the FMR1 gene

  • Thought to transport mRNAs

  • Expressed in brain, testes & ovaries

  • Males more severely affected

• X-linked Dominant Trait

• Results from expansion of the trinucleotide repeat CGG

• Three trinucleotide repeat states:

  • Normal 6 – 50 repeats

  • Premutation: 50 – 200 repeats

  • Affected: >200 repeats

Strand slippage in trinucleotide repeats

• triple repeat sequences are prone to changes in copy number during replication

• repeats usually involve G’s and C’s because they can form stranger base-pairs 

anticipation (dynamic mutation)

• disorders progressively worsen and/or appear earlier in future generations

• May depend on which parent the mutant allele comes from

• In Huntington disease, the TNRE is more likely to occur if inherited from the father

• In myotonic muscular dystrophy, the TNRE is more likely to occur if inherited from the mother (see Table 19.5)

• This suggests that TNRE can occur more frequently during oogenesis or spermatogenesis, depending on the gene involved

trinucleotide repeat expansion

• nature, location and number of TNRE varies in different diseases

• Tri-nucleotide repeats can happen in any part of a gene

• The repeat sequence is different for different diseases

• The number of repeats necessary to exhibit the disease can vary

Mutagens

• substances that induce mutations

• Almost any kind of mutation that can be induced by a mutagen can also occur spontaneously

• Mutagens bias the types of mutations that occur according to the type of damage to the DNA that they produce

alkylating agents

• donate an alkyl group to amino or keto groups in nucleotides to alter base-pairing affinity

• ethyl methane sulfonate (EMS): used for random mutagenesis

• prevents potential hydrogen bond, alters base pairing

base analogs

• can substitute for purine or pyrimidines during nucleic acid replication

• 5-bromouracil can pair with Adenine or Guanine

• used as cancer chemotherapy

intercalating agents 

• insert themselves into DNA - distorts mutations 

• often cause frameshift mutations

ultraviolet light

• purines and pyrimidines absorb UV radiation

• creates pyrimidine dimers that distort the DNA conformation in such a way that errors tend to be introduced during DNA replication

• extensive UV-induced dimerization is responsible for the killing effects of UV radiation on cells

oxidative damage

• oxidative reactions

• Reactive forms of oxygen

• Reactive Oxygen Species ROS

• Causes transversions

  • G pairs with A

  • GC → TA Transversion

• alters base pairing potential

ionizing radiation

• in the form of X rays, gamma rays, and cosmic rays are mutagenic

• Cause electrons to be ejected from atoms

• Directly damage bases, break phosphodiester bonds cause chromosomal aberrations

• Generates free radicals & Reactive Oxygen Species (ROS)

Ames Test

• biological assay to assess the mutagenic potential of chemical compounds

• Uses his- auxotrophic strains of the bacterium Salmonella typhimurium that have been selected for their increased sensitivity to mutagens

• Can reveal the presence of specific types of mutations depending on strain used

• Strains will require either point mutations or frameshifts to get reversion from his- to his+

• his- requires histidine

• his+ does not require histidine

  • Cell makes its own

• Number of his+ revertants is a measure of the severity of the mutagen

DNA repair 

• Direct repair

• Mismatch repair

• Base excision repair

• Nucleotide excision repair

• Double-strand break repair

direct repair

• photoreactivation repair: removes thymine dimers caused by UV light

  • enzyme involved is DNA photolyase

• alkylation damage can be repaired by alkyltransferase

  • enzyme catalyzes transfer of the alkyl group to itself

mmr

• bacterial DNA polymerase III is able to recognize and correct

  errors in replication, a process called proofreading

• corrects errors that remain after proofreading

  • Mismatches are detected

  • incorrect nucleotide is removed

  • correct nucleotide is inserted

• correct DNA strand is recognized based on DNA methylation of the parental strand

• old strand is hemimethylated, new strand is not

mmr proteins 

• mutS - binds to mismatch 

• mutH - binds to hemimethylated GATC

• mutL - binds to mutS and mutH 

• mutH makes one cut on the non-methylated DNA strand 

• cut strand is unwound by helicase 

• strand is degraded by an exonuclease

• dna polymerase synthesizes dna 

• liagse seals the nick in the dna 

lynch syndrome

• hereditary condition caused by germline mutations in mismatch repair genes

• individuals have an elevated risk of developing several cancers

excision repair

• three steps:

  • removal of the mutation by a nuclease

  • gap filling by dna polymerase

  • sealing the nick by dna ligase

Base Excision Repair (BER)

• form of postreplication repair 

• dna glycosylase recognizes erroneous base 

• cutting of the dna backbone at an AP site is done by an AP endonuclease 

Glycosylase

• removes altered bases from the DNA

• specific for different altered bases

• generates AP site

AP endonuclease

• single strand break/cut

• sugar removed by AP endonuclease

• gap filled by DNA polymerase

• nicked strand closed by dna ligase

Nucleotide Excision Repair (NER)

• Removes large distortions

• Initially described for repair of UV dimers

• 2 UvrA with UvrB scans the DNA for damage

• Upon recognition of damage UvrA leaves

• Recruits UvrC

• UvrBC complex cuts 5’ and 3’ of damage on damaged strand only

• Helicase removes damaged segment (13 nucleotides total)

• Gap is sealed by DNA polymerase I and ligase

Xeroderma pigmentosum

lost the ability to undergo nucleotide excision repair

Double-Strand Breaks