Molecular Basis of Change

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Molecular Basis of Change

• Core 218
• Biotechnology and Society
• Lecture number 8
• Spring 2007

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How Do Things Change?

• Gain new information
• Addition, incorporation, and use of new or
  foreign DNA.
• Mutation a change in the nucleotide sequence of
  the organisms genome
• Lose information
• Change information
• In English, a mutation is a change in genetic
  information.
• http//www-personal.ksu.edu/bethmont/mutdes.html

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Mutation, Why Study It?

• Mutations may be deleterious.
• Mutations may be advantageous (rarely) to an
  organism or its descendants.
• They are important to geneticists.
• To find out how a gene works, we must first shut
  off the gene
• The best way is to make variant (mutant) that
  lacks the ability to perform a process which we
  want to study.
• Use such mutants in complementation studies.
• Mutations fuel evolutionary change as it is a
  source of genetic variation.

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How Do Mutations Occur?

• Spontaneous mutation a change in an organisms
  DNA due to natural causes, i.e. DNA replication
  errors (about 3 in 10,000 to one in a million per
  gamete, per generation).
• Induced mutation a change in DNA that is caused
  by an external factor.
• Biological mutation a change in DNA that is
  caused by a biological or genetic factor.

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What Causes Mutations?

• Chemical mutagens chemicals that induce the DNA
  repair processes and increases the chance of
  errors in repair.
• Ionizing Radiation
• X-rays, radioactive materials, radon gas
• High energy radiation which causes increased
  reactivity of atoms in DNA and results in base
  changes and duplication/deletions.
• Exposure to extremely high-energy radiation has
  dire effects due to cell death.
• Ultraviolet radiation
• Absorbed by DNA bases which become more reactive.
• Usually Thymine dimerization which interferes
  with replication.
• Greatly enhances mutation rate.
• Biological entities - viral
• Genetic (inherent) - Transposable elements

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Chemical Mutagens

• Base analogs - structurally resemble purines and
  pyrimidines and may be incorporated into DNA in
  place of the normal bases during DNA replication.
• bromouracil (BU)--artificially created, resembles
  thymine (has Br atom instead of methyl group) and
  will be incorporated into DNA and pair with A
  like thymine. Higher likelihood for
  tautomerization to the enol form (BU)
• aminopurine --adenine analog which can pair with
  T or (less well) with C causes AT to GC or GC
  to AT transitions. Base analogs cause
  transitions, as do spontaneous tautomerization
  events.

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Chemical Mutagens

• Chemicals which alter structure and pairing
  properties of bases
• nitrous acid--formed by digestion of nitrites
  (preservatives) in foods. It causes C to U, meC
  to T, and A to hypoxanthine deaminations.
  Hypoxanthine in DNA pairs with C and causes
  transitions. Deamination by nitrous acid, like
  spontaneous deamination, causes transitions.
• nitrosoguanidine, methyl methanesulfonate, ethyl
  methanesulfonate--chemical mutagens that react
  with bases and add methyl or ethyl groups.
  Depending on the affected atom, the alkylated
  base may then degrade to yield a baseless site,
  which is mutagenic and recombinogenic, or mispair
  to result in mutations upon DNA replication.

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Chemical Mutagens

• Intercalating agents
• acridine orange, proflavin, ethidium bromide
  (used in labs as dyes and mutagens). All are
  flat, multiple ring molecules which interact with
  bases of DNA and insert between them. This
  insertion causes a "stretching" of the DNA duplex
  and the DNA polymerase is "fooled" into inserting
  an extra base opposite an intercalated molecule.
  The result is that intercalating agents cause
  frame shifts.
• Agents altering DNA structure - a "catch-all"
  category which includes a variety of different
  kinds of agents.
• large molecules which bind to bases in DNA and
  cause them to be noncoding--we refer to these as
  "bulky" lesions.
• agents causing intra- and inter-strand cross
  links (e. g. psoralens--found in some vegetables
  and used in treatments of some skin conditions)
• chemicals causing DNA strand breaks (e.g.
  peroxides).

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Spontaneous Mutation Rates
Spontaneous mutations are changes in DNA sequence
that result from errors in replication,
recombination of DNA, or by environmental
mutagens. Mutation rates vary relative to gene
size. Certain regions of DNA have hot spots
which have higher mutation rates.
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Mutation Sites (Targets)

• Germ line mutations mutation occurs in a germ
  cell (tissue that will form sex cells).
• Mutations are inherited (perpetuated from
  generation to generation).
• X-linked hemophilia mutation is thought to have
  occurred in the germ cells of Queen Victoria or
  one of her parents
• Somatic mutations mutation occurs in a cell in
  the developing somatic (body) tissue.
• Cells derived from progenitor cell are clonal
• Size of the mutant sector depends on when during
  development the mutation occurred i.e. the
  earlier in development, the larger the sector
• For diploids, only dominant mutations would be
  seen recessive mutations would only be seen if
  homozygous or heterozygous
• Mutations are not passed on to progeny

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Mutant Phenotypes

• Morphological mutations mutations that affect
  the visible properties of an organism e.g. shape,
  color, size.
• Lethal mutations the mutation affects the
  survival of the organism.
• Conditional mutations the mutant allele causes
  a mutant phenotype only under certain conditions
  (restrictive) but allows the wild type phenotype
  under other conditions (permissive).
• Biochemical mutations mutations that affect a
  biochemical function of the cell.
• Prototrophs (organisms that can grow on simple
  organic salts and a carbon source) when mutated
  can be Auxotrophs (biochemical mutants that
  require certain additional nutrients to grow).

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Mutant Phenotypes

• Loss-of-function mutations mutations that
  inactivate gene function.
• Recessive wild type (normal) allele (gene copy)
  is sufficient to produce the wild type phenotype
  in heterozygotes.
• Dominant the loss of function phenotype is
  expressed with a single mutant allele.
• Gain-of-function mutations the mutation confers
  some new function on the gene
• The new function is expressed in the
  heterozygote, so the mutation will likely act as
  a dominant allele

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Molecular Mechanisms

• Point mutations usually result from errors in
  replication of DNA or from the action of
  mutagens.
• Base substitutions
• Transitions purine for a different purine,
  pyrimidine for a different pyrimidine (e.g., A gt
  G, G gt A, T gt C, C gt T)
• Transversions purine for a pyrimidine, (e.g., A
  gt C, A gt T, G gt T, G gt C or pyramidine for a
  purine (e.g., T gt G, T gt A, C gt A, C gt G)
• Base additions or deletions 1 or more
  nucleotides added or deleted from both strands of
  the DNA

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Molecular Mechanisms

• Silent mutation codon specifies the same amino
  acid (e.g., Arg AGG gt CGG Arg, Phe UUU gt UUC
  Phe).
• Synonymous mutation codon specifies a different
  but functionally equivalent amino acid (e.g. Lys
  AAA gt AGA Arg, Ser UCU gt ACU Thr)
• Missense mutation codon specifies a different
  amino acid with a different (or non-) function.
• Nonsense mutation codon specifies protein chain
  termination i.e. stop codon (e.g., UAG, UAA, UGA).

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Molecular Mechanisms

• Frameshift mutation addition or deletion of
  base pairs that is not a multiple of 3.
• This changes the reading frame in protein coding
  regions, resulting in different residues from
  that point on and often premature termination
• Intragenic suppressor mutations frameshift of
  the opposite sign at a second site in gene
  resulting in restoration of reading frame.
• Second site missense mutation can also restore a
  functional interaction of the residues

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Biological Mutagens

• Transposons sometimes called jumping genes,
  are mobile segments of DNA that can move (hop)
  around to different locations in the genome of a
  cell. While doing so, they may cause mutations.

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5' GGCCAGTCACAATGG..400 nt..CCATTGTGACTGGCC
3'3' CCGGTCAGTGTTACC..400 nt..GGTAACACTGACCGG
5'
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Barbara McClintock started her career at the
University of Missouri. Used X-Rays to look for
genes in fruit flies, maize, and other
organisms. Found that in some cases, X-rays
actually broke chromosomes which then fused back
together in different ways. In the 1930s found
some stocks of her X-rayed maize plants whose
chromosomes spontaneous broke. This discovery
made her one of the great figures of maize
cytogenetics.
http//profiles.nlm.nih.gov/LL/B/B/Q/Q/
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About 44 of our genome is composed of
transposons or transposon-like repetitive
elements. There is about 2075 different
transposons. A single type can be almost 1800
locations.
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SO, HOW STABLE ARE YOU?