The Tryptophan Operon

1
The Tryptophan Operon
Figure 7.19
2
An inducible operon the lac operon

• Genes of the lac operon ?
• Normal conditions cell does not express lac
  genes
• If lactose is present in environment
• Activator
• Cell can metabolize lactose

3
The lac operon regulated synthesis of inducible
enzymes
3?
5?
Lactose absent, repressor active ?
4
The lac Operon Lactose
lac operon
mRNA 5?
mRNA 5'
Lactose present, repressor inactive ?
5
Control of the lac operon by catabolite activator
protein (CAP)
DNA
lacl
lacZ
ActiveCAP
cAMP
Inactive lac repressor
InactiveCAP
Lactose present, glucose scarce (cAMP level high)
?
6
The lac Operon Lactose Glucose
Lactose present, glucose present (cAMP level low)
?
7
The Lactose Operon
Figure 7.18
8

• Part 2 What happens when it doesnt work
  normally?

9
Mutations

• Types of mutations
• Effects of mutations
• Mutagens
• Radiation
• Chemicals
• DNA repair
• Selection of mutants

10
Reading Frames
? Deletion of a base can change the reading
frame ? Insertion of a base can change the
reading frame
11
Mutations Base-pair substitution
Wild type
A
U
G
A
A
G
U
U
U
G
G
C
U
A
A
mRNA
5?
3?
Protein
Lys
Met
Phe
Gly
Stop
Amino end
Carboxyl end
Base-pair substitution
Silent
U instead of C
A
U
G
A
A
G
U
U
U
G
G
U
U
A
A
Lys
Met
Phe
Gly
Stop
Missense
A instead of G
A
A
A
U
G
A
A
G
U
U
U
A
G
U
U
Lys
Met
Phe
Ser
Stop
Nonsense
U instead of A
G
A
A
U
U
G
G
G
U
A
U
U
U
C
A
Met
Stop
12
Mutations Base-pair insertion/ deletion
13
Effects of Mutation
Frameshift Mutations
Point Mutations
Figure 7.20
14
A point mutation causes sickle-cell disease
DNA mutant strand has an A wild-type strand has
a T.
Mutant hemoglobin DNA
Wild-type hemoglobin DNA
3?
5?
3?
5?
T
T
C
A
T
C
mRNA
mRNA
mRNA mutant has a U instead of an A in one
codon.
G
A
A
U
A
G
5?
3?
5?
3?
Protein mutant (sickle-cell) hemoglobin has a
valine (Val) instead of a glutamic acid (Glu).
Normal hemoglobin
Sickle-cell hemoglobin
Val
Glu
15
Types of Point Mutations and Effects
16
Radiation

• Ultraviolet Radiation (UV)
• covalent bonding
  between adjacent thymine bases
• and inhibition
  of replication and transcription
• X-Rays
• Single-strand, double-strand
• Base alterations

Why UV light is bad for DNA
17
Chemical Mutagens

• Chemical treatment that alters
• Modification by alkylating agents
• Addition of chemical groups (alkyl groups) onto
  bases
• ex nitrosoguanidine formerly used to treat
  cancer

18
Chemical Mutagens Nucleotide Analogs

• Compounds that resemble bases ?
• H-bonding properties are different ?

19
Chemical Mutagens Frameshift Mutagens

• Planar, ring molecules ?
• Results in addition of extra nucleotides
• ex acridine, ethidium bromide

20
Repair of Damaged DNA

• 24 hrs the DNA of every cell in your body is
  damaged 10,000 times!
• Unrepaired damage ?
• SOS repair
• Mechanism to bypass damaged DNA
• SOS ?
• Only found in
• Repair of thymine dimers
• Base-excision repair
• Mismatch Repair

21
DNA Repair repair of thymine dimers
thymine dimer is
broken using energy from visible light
thymine dimer is removed and
replaced
Light repair
Dark repair
22
DNA Repair
Base-excision repair
Mismatch repair
23
Genetics Vocabulary!

• properties of a
  cell determined by DNA composition
• characteristics
  displayed by an organism
• Bacteria are
• Humans are
• change in DNA base
  sequence from wild-type (normal)
• mutant that
  requires a growth factor
• cells that
  grow in absence of added growth factors

24
Conventions standard notations

• If mutation alters a gene for producing
  tryptophan ?
• Cells requiring tryptophan
• Cells that grow without added tryptophan
• Cells that carry resistance to antibiotic
  streptomycin
• Cells that are sensitive to streptomycin
• If no indication cells are assumed to be

25
Selection Positive (Direct) Selection for Mutants
26
Selection Negative (Indirect) Selection for
Mutants

• Replica plating
• Useful for negative selection of
  mutants
• ex Trp- mutant
• Parent grows
• Mutant grows

27
Types of Mutants

• mutation in
  essential gene that cannot be overcome
• 
  essential process only functions under certain
  conditions
• 
  mutant essential process only functions at
  certain temperatures
• ex Some protein function at 25º C but none at
  37º C

28
Genetic recombination and transfer

• Transformation
• Transduction
• Conjugation
• Transposition

29
Griffiths Experiments - Gene Transfer
1930s Frederick Griffith Discovered genetic
transformation
30
Gene Transfer

• Genetic material can be transferred from one
  individual to another
• 
  acquisition of genes from another individual of
  the same generation
• Ex
  (genetic transformation) - bacteria can
  take up free DNA from environment
• 
  acquisition of genes from a parent

31
Transduction
32
Bacterial Conjugation
Donor cell attaches to a recipient cell with its
pilus. The pilus draws the cells together.
The cells contact one another.
The plasmid replicates one of its strands and
transfers the daughter strand to the recipient.
The recipient simultaneously synthesizes a
complementary strand to become an F cell.
33
Bacterial Conjugation
Figure 7.31
34
Conjugation Hfr cells
35
Natural mechanisms of horizontal gene transfer in
bacteria
36
Transposable Elements

• DNA segments
  that move from one site to another in DNA
  molecules
• Insertions can disrupt gene function
• Can carry beneficial genes
• Ex

Barbara McClintock
37
Transposons
38
Why is gene transfer important in bacteria?

• Provides microbes with new genetic information ?
  
• Gene transfer allows many genes to be transferred
  simultaneously ?

39
Mechanisms of Gene Transfer

• 1. DNA-mediated transformation
• 2. Transduction
• Generalized transduction
• Specialized transduction
• 3. Conjugation
• F-plasmid transfer
• F- and F cells
• Chromosomal transfer
• Hfr cells
• 4. Transposition