CO 06
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Four requirements for DNA
to be genetic material
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Must carry information
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Must replicate
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DNA replication
Must allow for information to change
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Cracking the genetic code
Mutation
Must govern the expression of the phenotype
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Gene function
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DNA stores information in the sequence of its bases
•Much of DNA’s sequence-specific information is accessible only when the double
helix is unwound
•Proteins read the DNA sequence of nucleotides as the DNA helix unwinds.
Proteins can either bind to a DNA sequence, or initiate the copying of it.
•Human genome is believed to be 250 million nucleotides long. Four possible
nucleotides. Thus 4250,000,000 possible sequences in the human genome.
•An average single coding gene sequence might be about 10,000 bases long.
Thus, 410,000 possibilities for an average gene.
•Some genetic information is accessible even in intact, double-stranded DNA
molecules
•Some proteins recognize the base sequence of DNA without unwinding it.
•One example is a restriction enzyme.
3
Some viruses use RNA as the
repository of genetic information
Fig. 6.13
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Mutations: key tool in understanding
biological function
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What mutations are
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Mutations and gene structure
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Experiments using mutations demonstrate a gene is a discrete
region of DNA
Mutations and gene function
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How often mutations occur
What events cause mutations
How mutations affect survival and evolution
Genes encode proteins by directing assembly of amino acids
How do genotypes correlate with phenotypes?
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Phenotype depends on structure and amount of protein
Mutations alter genes instructions for producing proteins
structure and function, and consequently phenotype
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Mutations are heritable changes in base sequences that
modify the information content of DNA
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Substitution – base is replaced by one of the other
three bases
Deletion – block of one or more DNA pairs is lost
Insertion – block of one or more DNA pairs is
added
Inversion 1800 rotation of piece of DNA
Reciprocal translocation – parts of
nonhomologous chromosomes change places
Chromosomal rearrangements – affect many
genes at one time
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Fig. 7.2
7
Spontaneous mutations influencing
phenotype occur at a very low rate
Mutation rates from wild-type to recessive alleles for five coat color
genes in mice
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Fig. 7.3 b
Are mutations spontaneous or
induced?
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Most mutations are spontaneous.
Luria and Delbruck experiments - a simple
way to tell is mutations are spontaneous or
if they are induced by a mutagenic agent
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Fig. 7.4
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Replica plating verifies preexisting
mutations
Fig. 7.5 a
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Fig. 7.5b
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Interpretation of Luria-Delbruck fluctuation
experiment and replica plating
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Bacterial resistance arises from mutations
that exist before exposure to bacteriocide
After exposure to bacteriocide, the
bacteriocide becomes a selective agent
killing the nonresistant cells, allowing only
the preexisting resistant cells to survive.
Mutations do not arise in particular genes
as a direct response to environmental
change
Mutations occur randomly at any time
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Mistakes during replication alter
genetic information
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Errors during replication are exceedingly
rare, less than once in 109 base pairs
Proofreading enzymes correct errors made
during replication
DNA polymerase has 3’ – 5’ exonuclease activity
which recognizes mismatched bases and excises
it
 In bacteria, methyl-directed mismatch repair
finds errors on newly synthesized strands and
corrects them
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DNA polymerase proofreading
Fig. 7.8
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Methyldirected
mismatch
repair
Fig. 7.9
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Chemical and Physical agents cause
mutations
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Hydrolysis of a purine
base, A or G occurs 1000
times an hour in every cell
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Deamination removes –
NH2 group. Can change
C to U, inducing a
substitution to and A-T
base pair after replication
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X rays break the
DNA backbone
UV light produces
thymine dimers
Fig. 7.6 c, d
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Oxidation from free radicals formed by irradiation
damages individual bases
Fig. 7.6 e
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Repair enzymes fix errors created by
mutation
Excision repair
enzymes
release
damaged
regions of
DNA. Repair
is then
completed by
DNA
polymerase
and DNA ligase
Fig. 7.7a
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Unequal crossing over creates one
homologous chromosome with a duplication
and the other with a deletion
7.10 a
21
Trinucleotide repeat in people with
fragile X syndrom
Fig. A, B(2) Genetics and
Society
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Trinucleotide instability causes
mutations
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FMR-1 genes in
unaffected people
have fewer than
50 CGG repeats.
Unstable
premutation
alleles have
between 50 and
200 repeats.
Disease causing
alleles have > 200
CGG repeats.
Fig. B(1) Genetics and Society
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Mutagens induce mutations
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Mutagens can be used to increase mutation
rates
H. J. Muller – first discovered that X rays
increase mutation rate in fruitflies
Exposed male Drosophila to large doses of X
rays
 Mated males to females with balancer X
chromosome (dominant Bar eyed mutation and
multiple inversions)
 Could assay more than 1000 genes at once on
the X chromosome
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Muller’s experiment
Fig. 7.11
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Mutagens increase mutation rate
using different mechanisms
Fig. 7.12a
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Fig. 7.12 b
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Fig. 7.12 c
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Consequences of mutations
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Germ line mutations – passed on to next
generation and affect the evolution of
species
Somatic mutations – affect the survival of
an individual
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Cell cycle mutations may lead to cancer
Because of potential harmful affects of
mutagens to individuals, tests have been
developed to identify carcinogens
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The Ames test
for carcinogens
using hismutants of
Salmonella
typhimurium
Fig. 7.13
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What mutations tell us about gene
structure
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Complementation testing tells us whether two
mutations are in the same or different genes
Seymour Benzer’s phage experiments demonstrate
that a gene is a linear sequence of nucleotide pairs
that mutate independently and recombine with
each other, down to the adjacent-nucleotide level.
Some regions of chromosomes and even individual
bases mutate at a higher rate than others – hot
spots
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Complementation testing:
the cis-trans test identifies gene borders
Fig. 7.15 a
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Fig. 7.15 b,c
Five complementation groups (different genes) for eye color.
Recombination mapping demonstrates distance between genes and alleles.
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A gene is a linear sequence of
nucleotide pairs
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Seymore Benzer mid 1950s – 1960s
If a gene is a linear set of nucleotides,
recombination between homologous chromosomes
carrying different mutations within the same gene
should generate wild-type
 T4 phage as an experimental system – the rII gene
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Can examine a large number of progeny to detect rare
mutation events
 In the appropriate host, could allow only recombinant
phage to proliferate while parental phages died
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Hershey and Chase Waring blender
experiment
Fig. 6.5 a,b
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Fig. 6.5
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Benzer’s experimental procedure
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Generated 1612 spontaneous point mutations and
some deletions
Mapped location of deletions relative to one
another using recombination
Found approximate location of individual point
mutations by deletion mapping
Then performed recombination tests between all
point mutations known to lie in the same small
region of the chromosome
Result – fine structure map of the rII gene locus
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Working with T4 phage
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How recombination within a gene
could generate wild-type
Fig. 7.16
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Phenotpyic properties of T4 phage
Fig. 7.17 b
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Complementation test: are 2 mutations in the
same or different genes?
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Detecting recombination between
two mutations in the same gene
Fig. 7.17 d
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Deletions for rapid mapping of point
mutations to a region of the chromosome
Fig. 7.18 a
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Recombination
mapping to identify
the location of each
point mutation
within a small
region
Fig. 7.18 b
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Fine structure map of rII gene
region
Fig. 7.18 c
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Fig. 7a.p221
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What mutations tell us about gene
function
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One gene, one enzyme hypothesis: a gene contains
the information for producing a specific enzyme
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Beadle and Tatum use auxotrophic and prototrophic
strains of Neurospora to test hypothesis
Genes specify the identity and order of amino
acids in a polypeptide chain
The sequence of amino acids in a protein
determines its three-dimensional shape and
function
Some proteins contain more than one polypeptide
coded for by different genes
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Beadle and Tatum – One gene, one
enzyme
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1940s – isolated mutagen induced mutants that
disrupted synthesis of arginine, an amino acid
required for Neurospora growth
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Auxotroph – needs supplement to grow on minimal
media
Prototroph – wild-type that needs no supplement; can
synthesize all required growth factors
Recombination analysis located mutations in four
distinct regions of genome
Complementation tests showed each of four
regions correlated with different complementation
group (each was a different gene)
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Fig. 7.20 a
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Fig. 7.20 b
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Interpretation of Beadle and Tatum
experiments
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Each gene controls the synthesis of one of the
enzymes involved in catalyzing the conversion of
an intermediate into arginine.
These enzymes function sequentially.
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Genes specify the identity and order of
amino acids in a polypeptide chain
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Proteins are linear polymers of amino acids linked
by peptide bonds
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20 different amino acids are building blocks of proteins
NH2-CHR-COOH – carboxylic acid is acidic, amino
group is basic
R is the side chain that distinguishes each amino acid
Fig. 7.21 a
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R is the side group that distinguishes each
amino acid
Fig. 7.21 b
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Fig. 7.21 b
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N terminus of a protein contains a free amino group
C terminus of protein contains a free carboxylic acid group
Fig. 7.21 c
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Fig. 7.22
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Sequence of amino acids determine a proteins
primary, secondary, and tertiary structure
Fig. 7.23
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Some proteins are multimeric, containing subunits
composed of more than one polypeptide
Fig. 7.24
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Dominance relations between alleles depend on the
relation between protein function and phenotype
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Alleles that produce nonfunctional proteins are usually recessive
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Null mutations – prevent synthesis of protein or promote synthesis of
protein incapable of carrying out any function
Hypomorphic mutations – produce much less of a protein or a protein
with weak but detectable function; usually detectable only in
homozygotes
Incomplete dominance – phenotype varies in proportion to
amount of protein
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Hypermorphic mutations – produces more protein or same amount of a
more effective protein
Dominant negative – produces a subunit of a protein that blocks the
activity of other subunits
Neomorphic mutations – generate a novel phenotype; example is ectopic
expression where protein is produced outside of its normal place or time
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Fig. 6.17b
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Fig. 6.17c
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Fig. 6.17d
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Fig. 6.17e
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Fig. 6.17f
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Fig. 6.18abc
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Fig. 6.18def
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Fig. 6.19
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Fig. 6.20ab
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Fig. 6.20c
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Fig. 6.21
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Fig. 6.22a
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Fig. 6.22b
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Fig. 6.22c
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Fig. 6.22d
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Fig. 6.22e
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Fig. 6.22f
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Fig. 6.22g
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Fig. 6.22h
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