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Slide 1 - Evergreen Archives

VIEWS: 2 PAGES: 80

  • pg 1
									CO 06




        1
Four requirements for DNA
   to be genetic material
   Must carry information
       Cracking the genetic code
   Must replicate
       DNA replication
   Must allow for information to change
       Mutation
   Must govern the expression of the phenotype
       Gene function


                                                  2
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
                                                4
          Mutations: key tool in understanding
                        biological function
   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
                                                                    5
Mutations are heritable changes in base sequences that
       modify the information content of DNA

    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

                                                           6
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                            8
Fig. 7.3 b
      Are mutations spontaneous or
               induced?

   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




                                                  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 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
                                                    13
    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

                                                   14
       DNA polymerase proofreading




Fig. 7.8
                                     15
     Methyl-
     directed
     mismatch
      repair


Fig. 7.9
                16
    Chemical and Physical agents cause
               mutations
   Hydrolysis of a purine           Deamination removes –
    base, A or G occurs 1000          NH2 group. Can change
    times an hour in every cell       C to U, inducing a
                                      substitution to and A-T
                                      base pair after replication




                                                               17
        X rays break the
         DNA backbone




       UV light produces
        thymine dimers




Fig. 7.6 c, d

                            18
             Oxidation from free radicals formed by irradiation
                         damages individual bases




Fig. 7.6 e
                                                                  19
 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
                                          20
Fig. 7.7a
        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                 22
Society
      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 > 200
    CGG repeats.

                         Fig. B(1) Genetics and Society   23
        Mutagens induce mutations
   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
                                                  24
            Muller’s experiment




Fig. 7.11
                                  25
   Mutagens increase mutation rate
    using different mechanisms




Fig. 7.12a                           26
27
              28
Fig. 7.12 b
Fig. 7.12 c   29
        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

                                                  30
 The Ames test
for carcinogens
    using his-
   mutants of
   Salmonella
  typhimurium




Fig. 7.13     31
    What mutations tell us about gene
              structure
   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


                                                       32
              Complementation testing:
    the cis-trans test identifies gene borders




Fig. 7.15 a
                                                 33
Fig. 7.15 b,c



          Five complementation groups (different genes) for eye color.
     Recombination mapping demonstrates distance between genes and alleles.
                                                                              34
        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 – the rII gene
         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

                                                             35
  Hershey and Chase Waring blender
             experiment




Fig. 6.5 a,b

                                 36
Fig. 6.5




           37
Benzer’s 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

                                                        38
Working with T4 phage




                        39
  How recombination within a gene
     could generate wild-type




Fig. 7.16                           40
    Phenotpyic properties of T4 phage




Fig. 7.17 b                             41
Complementation test: are 2 mutations in the
        same or different genes?




                                           42
Detecting recombination between
 two mutations in the same gene




             Fig. 7.17 d
                                  43
     Deletions for rapid mapping of point
    mutations to a region of the chromosome




Fig. 7.18 a                                   44
         Recombination
       mapping to identify
       the location of each
         point mutation
          within a small
              region



Fig. 7.18 b             45
              Fine structure map of rII gene
                          region




Fig. 7.18 c
                                               46
Fig. 7a.p221




               47
    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


                                                            48
    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)

                                                               49
Fig. 7.20 a
              50
Fig. 7.20 b
              51
Interpretation of Beadle and Tatum
            experiments

   Each gene controls the synthesis of one of the
    enzymes involved in catalyzing the conversion of
    an intermediate into arginine.
   These enzymes function sequentially.




                                                       52
   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
                                                                         53
R is the side group that distinguishes each
                amino acid               Fig. 7.21 b




                                                  54
55
Fig. 7.21 b
              56
N terminus of a protein contains a free amino group
C terminus of protein contains a free carboxylic acid group




     Fig. 7.21 c                                              57
Fig. 7.22




            58
Sequence of amino acids determine a proteins
 primary, secondary, and tertiary structure




                                     Fig. 7.23
                                             59
Some proteins are multimeric, containing subunits
    composed of more than one polypeptide




                                           Fig. 7.24
                                                 60
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

                                                                            61
Fig. 6.17b




             62
Fig. 6.17c




             63
Fig. 6.17d




             64
Fig. 6.17e




             65
Fig. 6.17f




             66
Fig. 6.18abc




               67
Fig. 6.18def




               68
Fig. 6.19




            69
Fig. 6.20ab




              70
Fig. 6.20c




             71
Fig. 6.21




            72
Fig. 6.22a




             73
Fig. 6.22b




             74
Fig. 6.22c




             75
Fig. 6.22d




             76
Fig. 6.22e




             77
Fig. 6.22f




             78
Fig. 6.22g




             79
Fig. 6.22h




             80

								
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