GENE MUTATION AND DNA REPAIR

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GENE MUTATIONAND DNA REPAIR

• Chapter 16

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GENETIC MATERIAL

• DNA
• Primary function permanent storage of information
• Does not normally change
• Mutations do occur

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MUTATIONS

• Mutation
• Heritable change in the genetic material
• Permanent structural change of DNA
• Alteration can be passed on to daughter cells
• Mutations in reproductive cells can be passed to
  offspring

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MUTATIONS

• Mutations
• Provide allelic variation
• Ultimate source of genetic variation
• Foundation for evolutionary change
• Various phenotypic effects
• Neutral
• Harmful
• Beneficial

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MUTATIONS

• Mutations
• Most mutations are neutral
• More likely to be harmful than beneficial to the
  individual
• More likely to disrupt function than improve
  function

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MUTATIONS

• Mutations
• Many inherited diseases result from mutated genes
• Diseases such as various cancers can be caused by
  environmental agents known to cause DNA
  mutations
• Mutagens

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MODEL ORGANISMS

• Much of our understanding of mutations is a
  result of the study of model organisms
• e.g., Bacteria, yeast, Drosophila, etc.
• Amenable to analysis
• Short generation time, numerous offspring, etc.
• Often exposed to mutagenic environmental agents
• Effects of mutations are studied

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TYPES OF MUTATIONS

• Types of mutations
• Chromosome mutations
• Changes in chromosome structure
• Genome mutations
• Changes in chromosome number
• Single-gene mutations
• Relatively small changes in DNA structure
• Occur within a particular gene
• Focus of study in this chapter

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TYPES OF MUTATIONS

• Mutations involve the permanent alteration of a
  DNA sequence
• Alteration of base sequence
• Removal or addition of one or more nucleotides

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MUTATIONS

• Point mutations
• Change in a single base pair within the DNA
• Two main types of point mutations
• Base substitutions
• Transition
• Transversion
• Small deletions or insertions

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MUTATIONS

• Two types of base substitutions
• Transition
• Pyrimidine changed to another pyrimidine
• e.g., C ? T
• Purine changed to another purine
• e.g., A ? G
• Transversion
• Purines and pyrimidines are interchanged
• e.g., A ? C
• More rare than transitions

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EFFECTS OF MUTATIONS

• Mutations within the coding sequence of a gene
  can have various effects on the encoded
  polypeptides amino acid sequence
• Silent mutations
• Missense mutations
• Included neutral mutations
• Nonsense mutations
• Frameshift mutations

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EFFECTS OF MUTATIONS

• Silent mutations
• Amino acid sequence is not altered
• e.g., CCC ? CCG (pro ? pro)
• Genetic code is degenerate
• Alterations of the third base of a codon often do
  not alter the encoded amino acid
• Phenotype is not affected

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EFFECTS OF MUTATIONS

• Missense mutations
• Amino acid sequence is altered
• e.g., GAA ?GTA (glu ? val)
• Phenotype may be affected

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EFFECTS OF MUTATIONS

• Neutral mutations
• Type of missense mutation
• Amino acid sequence is altered
• e.g., CTT ?ATT (leu ? ile)
• e.g., GAA ?GAC (glu ? asp)
• No detectable effect on protein function
• Missense mutations substituting an amino acid
  with a similar chemistry to the original is
  likely to be neutral

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EFFECTS OF MUTATIONS

• Nonsense mutations
• Normal codon is changed into a stop codon
• e.g., AAA ? AAG (lys ? stop)
• Translation is prematurely terminated
• Truncated polypeptide is formed
• Protein function is generally affected

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EFFECTS OF MUTATIONS
18
EFFECTS OF MUTATIONS
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EFFECTS OF MUTATIONS

• Mutations occasionally produce a polypeptide with
  an enhanced ability to function
• Relatively rare
• May result in an organism with a greater
  likelihood to survive and reproduce
• Natural selection may increase the frequency of
  this mutation in the population

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MUTATION TYPES

• Genetic terms to describe mutations
• Wild-type
• Relatively common genotype
• Generally the most common allele
• Variant
• Mutant allele altering an organisms phenotype
• Forward mutation
• Changes wild-type allele into something else
• Reverse mutation
• Reversion
• Restores wild-type allele

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MUTATION TYPES

• Genetic terms to describe mutations
• Deleterious mutation
• Decreases an organisms chance of survival
• Lethal mutation
• Results in the death of an organism
• Extreme example of a deleterious mutation
• Conditional mutants
• Affect the phenotype only under a defined set of
  conditions
• e.g., Temperature-sensitive (ts) mutants

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MUTATION TYPES

• Genetic terms to describe mutations
• Suppressor mutation
• Second mutation that restores the wild-type
  phenotype
• Intragenic suppressor
• Secondary mutation in the same gene as the first
  mutation
• Differs from a reversion
• Second mutation is at a different site than the
  first
• Intergenic suppressor
• Secondary mutation in a different gene than the
  first mutation

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MUTATION TYPES

• Two general types of intergenic suppressors
• Those involving an ability to defy the genetic
  code
• Those involving a mutant structural gene

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MUTATION TYPES

• Intergenic suppressor mutations involving an
  ability to defy the genetic code
• e.g., tRNA mutations
• Altered anticodon region
• e.g., Recognize a stop codon
• May suppress a nonsense mutation in a gene
• May also suppress stop codons in normal genes

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MUTATION TYPES

• Intergenic suppressors involving a mutant
  structural gene
• Usually involve altered expression of one gene
  that compensates for a loss-of-function mutation
  affecting another gene
• Second gene may take over the functional role of
  the first
• May involve proteins participating in a common
  cellular function
• Sometimes involve mutations in genetic regulatory
  proteins
• e.g., Transcription factors activating other
  genes that can compensate for the mutation in the
  first gene

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MUTATION TYPES

• Mutations occurring outside of coding sequences
  can influence gene expression
• Mutations may alter the core promoter sequence
• Up promoter mutations
• Mutant promoter becomes more like the consensus
  sequence
• Rate of transcription may be increased
• Down promoter mutations
• Mutant promoter becomes less like the consensus
  sequence
• Affinity for regulatory factors is decreased
• Rate of transcription may be decreased

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MUTATION TYPES

• Mutations occurring outside of coding sequences
  can influence gene expression
• Mutations may alter other regulatory sequences
• lacOC mutations prevent binding of the lac
  repressor
• Lac operon is constituently expressed, even in
  the absence of lactose
• Such expression is wasteful
• Such mutants are at a selective disadvantage

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MUTATION TYPES

• Mutations occurring outside of coding sequences
  can influence gene expression
• Mutations may alter splice junctions
• Altered order and/or number of exons in the mRNA

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MUTATION TYPES

• Mutations occurring outside of coding sequences
  can influence gene expression
• Mutations may affect an untranslated region of
  mRNA
• 5- or 3-UTR
• May affect mRNA stability
• May affect the ability of the mRNA to be
  translated

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MUTATION TYPES
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TRINUCLEOTIDE REPEATS

• DNA trinucleotide repeats
• Three nucleotide sequences repeated in tandem
• e.g., CAGCAGCAGCAGCAGCAG
• Generally transmitted normally from parent to
  offspring without mutation

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TRINUCLEOTIDE REPEATS

• Trinucleotide repeat expansion (TNRE)
• Number of repeats can readily increase from one
  generation to the next
• Cause of several human genetic diseases
• Length of a repeat has increased above a certain
  critical size
• Becomes prone to frequent expansion

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TRINUCLEOTIDE REPEATS

• TNRE disorders
• Fragile X syndrome (FRAXA)
• FRAXE mental retardation
• Myotonic muscular dystrophy (DM)
• Spinal and bulbar muscular atrophy (SBMA)
• Huntington disease (HD)
• Spinocerebellar ataxia (SCA1)

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TRINUCLEOTIDE REPEATS

• TNRE disorders

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TRINUCLEOTIDE REPEATS

• TNRE disorders
• Expansion may be within a coding sequence of a
  gene
• Most expansions are of a CAG repeat
• Encoded proteins possess long tracts of glutamine
• CAG encodes a glutamine codon
• Presence of glutamine tracts causes aggregation
  of the proteins
• Aggregation is correlated with the progression of
  the disease

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TRINUCLEOTIDE REPEATS

• TNRE disorders
• Expansion may be in a noncoding region of a gene
• Two fragile X syndromes
• Repeat produces CpG islands that become
  methylated
• Methylation can lead to chromosome compaction
• Can silence gene transcription
• Myotonic muscular dystrophy
• Expansions may cause abnormal changes in RNA
  structure

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TRINUCLEOTIDE REPEATS

• TNRE disorders
• Severity of the disease tends to worsen in future
  generations
• Anticipation
• Severity of the disease depends on the parent
  from whom it was inherited
• e.g., In Huntingdon disease, TNRE likely to occur
  if mutation gene is inherited from the father
• e.g., In myotonic muscular dystrophy, TNRE likely
  to occur if mutation gene is inherited from the
  mother

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TRINUCLEOTIDE REPEATS

• TNRE disorders

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TRINUCLEOTIDE REPEATS

• TNRE disorders
• Cause of TNRE is not well understood
• Trinucleotide repeat may produce alterations in
  DNA structure
• e.g., Stem-loop formation
• May lead to errors in DNA replication
• TNRE within certain genes alters gene expression
• Disease symptoms are produced

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CHROMOSOME STRUCTURE

• Altered chromosome structure can alter gene
  expression
• Inversions and translocations commonly have no
  obvious phenotypic effects
• Phenotypic effects sometimes occur
• Position effect

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CHROMOSOME STRUCTURE

• Altered chromosome structure can alter gene
  expression and phenotype
• Breakpoint may occur within a gene
• Expression of the gene is altered
• Breakpoint may occur near a gene
• Expression is altered when moved to a new
  location
• May be moved next to regulatory elements
  influencing the expression of the relocated gene
• i.e., Silencers or enhancers
• May reposition a gene from a euchromatic region
  to a highly condensed (heterochromatic) region
• Expression may be turned off

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CHROMOSOME STRUCTURE

• Altered chromosome structure can alter gene
  expression and phenotype
• An eye color gene relocated to a heterochromatic
  region can display altered expression
• Gene is sometimes inactivated
• Variegated phenotype results

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SOMATIC VS. GERM-LINE

• The timing of mutations in multicellular
  organisms plays an important role
• Mutations may occur in gametes or a fertilized
  egg
• Mutations may occur later in life
• Embryonic or adult stages
• Timing can affect
• The severity of the genetic effect
• The ability to be passed from parent to offspring

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SOMATIC VS. GERM-LINE

• Animals possess germ-line and somatic cells
• Germ-line cells
• Cells giving rise to gametes
• Somatic cells
• All cells of the body excluding the germ-line
  cells
• e.g., Muscle cells, nerve cells, etc.

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SOMATIC VS. GERM-LINE

• Germ-line cells
• Germ-line mutations can occur in gametes
• Germ-line mutations can occur in a precursor cell
  that produces gametes
• All cells in the resulting offspring will contain
  the mutation

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SOMATIC VS. GERM-LINE

• Somatic cells
• Somatic mutations in embryonic cells can result
  in patches of tissues containing the mutation
• Size of the patch depends on the timing of the
  mutation
• Individual is a genetic mosaic

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CAUSES OF MUTATIONS

• Two causes of mutations
• Spontaneous mutations
• Result from abnormalities in biological
  processes
• Underlying cause lies within the cell
• Induced mutations
• Caused by environmental agents
• Cause originates outside of the cell

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CAUSES OF MUTATIONS

• Causes of spontaneous mutations
• Abnormalities in crossing over
• Aberrant segregation of chromosomes during
  meiosis
• Mistakes by DNA polymerase during replication
• Alteration of DNA by chemical products of normal
  metabolic processes
• Integration of transposable elements
• Spontaneous changes in nucleotide structure

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CAUSES OF MUTATIONS

• Induced mutations are caused by mutagens
• Chemical substances or physical agents
  originating outside of the cell
• Enter the cell and then alter the DNA structure

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CAUSES OF MUTATIONS

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CAUSES OF MUTATIONS

• Spontaneous mutations are random events
• Not purposeful
• Mutations occur as a matter of chance
• Some individuals possess beneficial mutations
• Better adapted to their environment
• Increased chance of surviving and reproducing
• Natural selection results in differential
  reproductive success
• The frequency of such alleles increases in the
  population

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CAUSES OF MUTATIONS

• Salvador Luria and Max Delbruck
• T1 is a bacteriophage able to infect E coli
• A small percentage of bacteria are resistant to
  T1 infection
• Heritable trait
• tonr (T1 resistance)
• Is this resistance due to spontaneous mutations
  or due to a physiological adaptation?

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CAUSES OF MUTATIONS

• Salvador Luria and Max Delbruck
• The question
• Is T1 resistance due to spontaneous mutations or
  due to a physiological adaptation?

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CAUSES OF MUTATIONS

• Salvador Luria and Max Delbruck
• Two competing theories
• Adaptation theory
• Rate of adaptation should be relatively constant
• Depends on exposure to bacteriophage
• tonr cells should be a relatively constant
  proportion of the total bacterial population
• Spontaneous mutation theory
• Number of tonr cells is dependent on timing of
  mutation
• tonr mutation occurring early in proliferation ?
  many tonr mutants found
• tonr mutation occurring late in proliferation ?
  fewer tonr mutants found
• Predicts greater variation in the number of tonr
  cells present in different populations

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CAUSES OF MUTATIONS

• Salvador Luria and Max Delbruck
• The experiment
• Fluctuation test
• Grew T1-susceptible bacteria in a flask and in
  several individual tubes
• Plated onto media with T1 phage
• Counted number of tonr colonies

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CAUSES OF MUTATIONS

• Salvador Luria and Max Delbruck
• The results
• Even distribution of tonr colonies from large
  flask
• Great fluctuation in number of tonr colonies from
  small tubes

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CAUSES OF MUTATIONS

• Salvador Luria and Max Delbruck
• The conclusion
• Results are consistent with the spontaneous
  mutation theory
• Timing of the mutation during the growth of a
  culture greatly affects the number of mutant cells

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CAUSES OF MUTATIONS

• Joshua and Ester Lederberg (1950s)
• Interested in the relationship between mutation
  and the environmental conditions shat select for
  mutations
• Scientists were unsure of the relationship
• Two competing hypotheses
• Directed mutation hypothesis
• Some scientists still believed that selective
  conditions could promote specific mutations
• Random mutation theory
• Mutations occur at random
• Environmental factors affecting survival select
  for those possessing beneficial mutations

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CAUSES OF MUTATIONS

• Joshua and Ester Lederberg (1950s)
• Plated large number of bacteria onto a master
  plate
• Contained no selective agent
• Transferred colonies to secondary plates
  containing selective agent (T1 phage)
• Replica plating
• Only mutant cells would grow

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CAUSES OF MUTATIONS

• Joshua and Ester Lederberg (1950s)
• Mutants occupied the same locations on all
  secondary plates
• Indicated that mutations occurred randomly in
  colonies growing on the nonselective master
  plate
• Ransom mutation theory is supported

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CAUSES OF MUTATIONS

• Mutation rate
• Likelihood that a gene will be altered by a new
  mutation
• Expressed as the number of new mutations in a
  given gene per generation
• Generally 1/100,000 1/billion
• 10-5 10-9

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CAUSES OF MUTATIONS

• Mutation rate
• Mutation rate is not a constant number
• Can be increased by environmental mutagens
• Induced mutations can increase beyond frequency
  of spontaneous mutations
• Mutation rates vary extensively between species
• Even vary between strains of the same species

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CAUSES OF MUTATIONS

• Mutation rate
• Some genes mutate at a much higher rate than
  other genes
• Some genes are longer than others
• Some locations are more susceptible to mutation
• Even single genes possess mutation hot spots
• More likely to mutate than other regions

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CAUSES OF MUTATIONS

• Mutation frequency
• Number of mutant alleles of a given gene divided
  by the number of alleles within a population
• Timing of mutations influences mutation frequency
• Timing does not influence mutation rate
• Mutation frequency depends both on mutation rate
  and timing of mutations
• Natural selection and genetic drift can further
  increase mutation frequencies

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CAUSES OF MUTATIONS

• Spontaneous mutations Depurination
• Most common type of naturally occurring chemical
  change
• Reaction with water removes a purine (A or G)
  from the DNA
• Apurinic site

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CAUSES OF MUTATIONS

• Spontaneous mutations Depurination
• 10,000 purines lost per 20 hours at 37oC in a
  typical mammalian cell
• Rate of loss increased by agents causing certain
  base modification
• e.g., Attachment of alkyl (methyl, ethyl, etc.)
  groups
• Generally recognized by DNA repair enzymes
• Mutation may result if repair system fails

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CAUSES OF MUTATIONS

• Spontaneous mutations Deamination of cytosines
• Other bases are not readily deaminated
• Removal of an amino group from the cytosine base
• Uracil is produced
• DNA repair enzymes generally remove this base
• Uracil is recognized as an inappropriate base
• Mutation may result if repair system fails
• Uracil hydrogen bonds with A, not G

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CAUSES OF MUTATIONS

• Spontaneous mutations Deamination of cytosines
• Methylation of cytosine occurs in many eukaryotic
  species as well as prokaryotes
• Removal of an amino group from the 5-methyl
  cytosine produces thymine
• DNA repair enzymes cannot determine which is the
  incorrect base
• Hot spots for mutations are produced

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CAUSES OF MUTATIONS

• Spontaneous mutations Tautomeric shifts
• Common, stable form of T and G is the keto form
• Interconvert to an enol form at a low rate
• Common, stable form of A and C is the amino form
• Interconvert to an imino form at a low rate

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CAUSES OF MUTATIONS

• Spontaneous mutations Tautomeric shifts
• Enol and imino forms do not conform to normal
  base-pairing rules
• AC and GT base pairs are formed

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CAUSES OF MUTATIONS

• Spontaneous mutations Tautomeric shifts
• Tautomeric shifts immediately prior to DNA
  replication can cause mutations
• Resulting mismatch could be repaired
• Mutation may result if repair system fails

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CAUSES OF MUTATIONS

• Hermann Muller (1927)
• Showed that X rays can cause induced mutations
• Reasoned that a mutagenic agent might form
  defective alleles
• Experimental approach focused on formation and
  detection of X-linked genes in Drosophila
  melanogaster

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CAUSES OF MUTATIONS

• Hermann Muller (1927)
• The hypothesis
• Exposure to X rays will increase the rate of
  mutation

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CAUSES OF MUTATIONS

• Hermann Muller (1927)
• The materials
• Drosophila melanogaster strain with three genetic
  alterations of the X chromosome
• ClB chromosome
• C Large inversion preventing productive
  crossing over
• l Lethal recessive X-linked gene
• B Bar eye allele

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CAUSES OF MUTATIONS

• Hermann Muller (1927)
• The design
• Females with one ClB chromosome and one normal
  chromosome
• Cannot undergo crossovers in the C region
• Can only produce sons possessing the normal X
  chromosome
• Lethal mutations in the normal chromosome will
  prevent them from producing any sons

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CAUSES OF MUTATIONS

• Hermann Muller (1927)
• The experiment
• Exposed wild-type males to X rays
• May mutate the X chromosome in sperm
• Mated mutagenized males to females with the ClB
  chromosome
• Daughters with bar eyes were saved
• Mated to wild-type males
• Genders of offspring determined

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CAUSES OF MUTATIONS

• Hermann Muller (1927)
• The data

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CAUSES OF MUTATIONS

• Hermann Muller (1927)
• Interpreting the data
• Very few lethal mutations occurred in the
  untreated control
• Approximately 1 in 1,000
• Many more mutations occurred in the X ray-treated
  flies
• Nearly 100 times more
• X rays greatly increase the rate of X-linked
  recessive lethal mutations

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CAUSES OF MUTATIONS

• The public is concerned about mutagens for two
  important reasons
• Mutagenic agents are often involved in the
  development of human cancers
• Avoiding mutations that may have harmful effects
  on future offspring is desirable

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CAUSES OF MUTATIONS

• An enormous array of agents can act as mutagens
• Chemical agents and physical agents

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CAUSES OF MUTATIONS

• Certain non-mutagenic chemicals can be altered to
  a mutagenically active form after ingestion
• Cellular enzymes such as oxidases can activate
  some mutagens
• Certain foods contain chemicals acting as
  antioxidants
• Antioxidants may be able to counteract the
  effects of mutagens and lower cancer rates

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CAUSES OF MUTATIONS

• Mutagens alter DNA structure in various ways
• Nitrous acid (HNO3) replaces amino groups with
  keto groups
• -NH2 ? O
• Can change cytosine to uracil
• Pairs with A, not G
• Can change adenine to hypoxanthine
• Pairs with C, not T

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CAUSES OF MUTATIONS

• Mutagens alter DNA structure in various ways
• Alkylating agents covalently attach methyl or
  ethyl groups to bases
• e.g., Nitrogen mustards, ethyl methanesulfonate
  (EMS)
• Appropriate base pairing is disrupted

84
CAUSES OF MUTATIONS

• Mutagens alter DNA structure in various ways
• Some mutagens directly interfere with the DNA
  replication process
• e.g., Acridine dyes such as proflavin
• Flat, planar structures interchelate into the
  double helix
• Sandwich between adjacent base pairs
• Helical structure is distorted
• Single-nucleotide additions and deletions can
  result

85
CAUSES OF MUTATIONS

• Mutagens alter DNA structure in various ways
• Some mutagens are base analogs
• e.g., 2-aminopurine
• e.g., 5-bromouracil (5BU)
• Become incorporated into daughter strands during
  DNA replication

86
CAUSES OF MUTATIONS

• Mutagens alter DNA structure in various ways
• Some mutagens are base analogs
• 5-bromouracil (5BU) is a thymine analog
• Incorporated in place of thymine
• 5BU can base-pair with adenine
• Can tautomerize and base-pair with guanine at a
  relatively high rate
• AT ? A5BU ? G5BU ? GC
• Transition mutations occur

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CAUSES OF MUTATIONS

• Mutagens alter DNA structure in various ways
• DNA molecules are sensitive to physical agents
  such as radiation
• e.g., Ionizing radiation such as X rays and gamma
  rays
• Short wavelength and high energy
• Can penetrate deeply into biological materials
• Creates free radicals
• Chemically reactive molecules
• Free radicals alter DNA structure in a variety of
  ways
• Deletions, single nicks, cross-linking,
  chromosomal breaks

88
CAUSES OF MUTATIONS

• Mutagens alter DNA structure in various ways
• DNA molecules are sensitive to physical agents
  such as radiation
• e.g., Nonionizing radiation such as UV light
• Contains less energy
• Penetrates only the surface of material such as
  the skin
• Causes the formation of thymine dimers
• May be repaired through one of numerous repair
  systems
• May cause a mutation when that DNA strand is
  replicated

89
CAUSES OF MUTATIONS

• Many different kinds of testes can determine if
  an agent is mutagenic
• Ames test is commonly used
• Developed by Bruce Ames
• Uses his- strains of Salmonella typhimurium
• Mutation is due to a point mutation rendering an
  enzyme inactive
• Reversions can restore his phenotype
• Ames test monitors rate of reversion mutations

90
CAUSES OF MUTATIONS

• Ames test
• Suspected mutagen is mixed with rat liver extract
  and his- Salmonella typhimurium
• Rat liver extract provides cellular enzymes that
  may be required to activate a mutagen
• Bacteria are plated on minimal media
• his revertants can be detected
• Mutation frequency calculated
• Compared to control

91
DNA REPAIR

• Most mutations are deleterious
• DNA repair systems are vital to the survival
• Bacteria possess several different DNA repair
  systems
• Absence of a single system greatly increases
  mutation rate
• Mutator strains
• Humans defective in a single DNA repair system
  may manifest various disease symptoms
• e.g., Higher risk of skin cancer

92
DNA REPAIR

• Living cells contain several DNA repair systems
• Able to fix different types of DNA alterations