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Anatomy%20and%20Function%20of%20a%20Gene

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Title: Anatomy%20and%20Function%20of%20a%20Gene


1
Anatomy and Function of a Gene
  • Dissection through mutation

2
Outline of Chapter 7
  • What mutations are
  • How often mutations occur
  • What events cause mutations
  • How mutations affect survival and evolution
  • Mutations and gene structure
  • Experiments using mutations demonstrate a gene is
    a discrete region of DNA
  • Mutations and gene function
  • Genes encode proteins by directing assembly of
    amino acids
  • How do genotypes correlate with phenotypes?
  • Phenotype depends on structure and amount of
    protein
  • Mutations alter genes instructions for producing
    proteins structure and function, and consequently
    phenotype

3
Mutations Primary tools of genetic analysis
  • Mutations are heritable changes in base sequences
    that modify the information content of DNA
  • Forward mutation changes wild-type to different
    allele
  • Reverse mutation causes novel mutation to
    revert back to wild-type (reversion)

4
Classification of mutations by affect on DNA
molecule
  • 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 180o rotation of piece of DNA
  • Reciprocal translocation parts of nonhomologous
    chromosomes change places
  • Chromosomal rearrangements affect many genes at
    one time

5
Fig. 7.2
6
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
Fig. 7.3 b
7
General observations of mutation rates
  • Mutations affecting phenotype occur very rarely
  • Different genes mutate at different rates
  • Rate of forward mutation is almost always higher
    than rate of reverse mutation

8
Are mutations spontaneous or induced?
  • Most mutations are spontaneous.
  • Luria and Delbruck experiments - a simple way to
    tell if mutations are spontaneous or if they are
    induced by a mutagenic agent

9
Fig. 7.4
10
Replica plating verifies preexisting mutations
Fig. 7.5 a
11
Fig. 7.5b
12
Interpretation of Luria-Delbruck fluctuation
experiment and replica plating
  • Bacterial resistance arises from mutations that
    exist before exposure to bactericide
  • After exposure to bactericide, the bactericide
    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

13
Chemical and Physical agents cause mutations
  • Hydrolysis of a purine base, A or G occurs 1000
    times an hour in every cell
  • Deamination removes NH2 group. Can change C to
    U, inducing a substitution to and A-T base pair
    after replication

Fig. 7.6 a,b
14
  • X rays break the DNA backbone
  • UV light produces thymine dimers

Fig. 7.6 c, d
15
Oxidation from free radicals formed by
irradiation damages individual bases
Fig. 7.6 e
16
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
17
Mistakes during replication alter genetic
information
  • 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

18
DNA polymerase proofreading
Fig. 7.8
19
Methyl-directed mismatch repair
Fig. 7.9
20
Unequal crossing over creates one homologous
chromosome with a duplication and the other with
a deletion
7.10 a
21
Transposable elements move around the genome and
are not susceptible to excision or mismatch repair
Fig. 7.10 e
22
Trinucleotide instability causes mutations
  • 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 gt 200 CGG repeats.

Fig. B(1) Genetics and Society
23
Trinucleotide repeat in people with fragile X
syndrom
Fig. A, B(2) Genetics and Society
24
Mutagens induce mutations
  • Mutagens can be used to increase mutation rates
  • H. J. Muller first discovered that X rays
    increase mutation rate in fruit flies
  • 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

25
Mullers experiment
Fig. 7.11
26
Mutagens increase mutation rate using different
mechanisms
Fig. 7.12a
27
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28
Fig. 7.12 b
29
Fig. 7.12 c
30
Consequences of mutations
  • Germ line mutations passed on to next
    generation and affect the evolution of species
  • Somatic mutations affect the survival of an
    individual
  • Cell cycle mutations may lead to cancer
  • Because of potential harmful affects of mutagens
    to individuals, tests have been developed to
    identify carcinogens

31
The Ames test for carcinogens using his- mutants
of Salmonella typhimurium
Fig. 7.13
32
What mutations tell us about gene structure
  • Complementation testing tells us whether two
    mutations are in the same or different genes
  • Benzers experiments demonstrate that a gene is a
    linear sequence of nucleotide pairs that mutate
    independently and recombine with each other
  • Some regions of chromosomes mutate at a higher
    rate than others hot spots

33
Complementation testing
Fig. 7.15 a
34
Fig. 7.15 b,c
Five complementation groups (different genes) for
eye color. Recombination mapping demonstrates
distance between genes and alleles.
35
A gene is a linear sequence of nucleotide pairs
  • 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
  • Can examine a large number of progeny to detect
    rare mutation events
  • Could allow only recombinant phage to proliferate
    while parental phages died

36
Benzers experimental procedure
  • 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

37
How recombination within a gene could generate
wild-type
Fig. 7.16
38
Working with T4 phage
39
Phenotpyic properties of T4 phage
Fig. 7.17 b
40
Complementation test to for mutations in
different genes
41
Detecting recombination between two mutations in
the same gene
Fig. 7.17 d
42
Deletions for rapid mapping of point mutations to
a region of the chromosome
Fig. 7.18 a
43
Recombination mapping to identify the location of
each point mutation within a small region
Fig. 7.18 b
44
Fine structure map of rII gene region
Fig. 7.18 c
45
What mutations tell us about gene function
  • One gene, one enzyme hypothesis a gene contains
    the information for producing a specific enzyme
  • 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

46
Beadle and Tatum One gene, one enzyme
  • 1940s isolated mutagen induced mutants that
    disrupted synthesis of arginine, an amino acid
    required for Neurospora growth
  • 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)

47
Fig. 7.20 a
48
Fig. 7.20 b
49
Interpretation of Beadle and Tatum experiments
  • Each gene controls the synthesis of an enzyme
    involved in catalyzing the conversion of an
    intermediate into arginine

50
Genes specify the identity and order of amino
acids in a polypeptide chain
  • Proteins are linear polymers of amino acids
    linked by peptide bonds
  • 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
51
R is the side group that distinguishes each amino
acid
Fig. 7.21 b
52
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53
Fig. 7.21 b
54
N terminus of a protein contains a free amino
group C terminus of protein contains a free
carboxylic acid group
Fig. 7.21 c
55
Genes specify the amino acid sequence of a
polypeptide example, sickle cell anemia
Mutant b chain of hemoglobin form aggregates that
cause red blood cells to sickle
Fig. 7.22 a
56
Sequence of amino acids determine a proteins
primary, secondary, and tertiary structure
Fig. 7.23
57
Some proteins are multimeric, containing subunits
composed of more than one polypeptide
Fig. 7.24
58
How do genotypes and phenotypes correlate?
  • Alteration of amino acid composition of a protein
  • Alteration of the amount of normal protein
    produced
  • Changes in different amino acids at different
    positions have different effects
  • Proteins have active sites and sites involved in
    shape or structure

59
Dominance relations between alleles depend on the
relation between protein function and phenotype
  • Alleles that produce nonfunctional proteins are
    usually recessive
  • 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
  • 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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