Transcript Mutation - MCCC Faculty & Staff Web Pages

Mutation, DNA Repair and
Recombination
BIT 220
Chapter 14
Mutation
• Heritable change in
genetic material
– change in gene
– change in chromo #
– change in chromo
structure
• Germinal Mutation
– may be passed to
progeny
• Somatic Mutation
– NOT passed to
progeny offspring
What causes mutation?
• SPONTANEOUS
– No known cause
– maybe be indeed a
causative agent, but
has not been identified
• INDUCED
– Caused by MUTAGEN
• (physical or chemical
agents which alter
DNA)
•
•
•
•
nicotine
UV light
drugs
ionizing radiation
Phenotypic Effects
• Neutral Mutation- NO Effect on phenotype
» ISOALLELES
• Null Allele - No functional protein
• Recessive Lethal - deadly null mutation in both
alleles
Conditional Lethal Mutations
Define:
Lethal in one environment (restrictive condition)
Viable in second environment (permissive condition)
1. Auxotrophs Mutants - can’t synthesize metabolite
2. Temperature-Sensitive mutants - grow in one T, not another T
3. Suppressor-sensitive mutants - will grow in presence of suppressor;
suppressor protein compensates for mutation
Replica Plating
• Figure 14.3
• Non-Selective Plates -no antibiotic
• Selective plates- antibiotic in medium
• Mutation does NOT occur in response to
selective agent
Frameshift Mutations
Base pair addition or deletion
•alter reading frame
•always result in non-functional protein
Forward and Reverse Mutations
• Figure 14.4
• Forward: wild-type to mutant phenotype
• Reverse: 2nd mutation restores earlier
phenotype; reverse occurs by back mutation
or suppressor mutation
• Summary of mutation process: Figure 14.7
• Example (sickle-cell) Figure 14.8
Transposons
Figure
14.24
Pieces of DNA that can move from one location of genome to another
No specific target site
Effects:
•help bacteria to adapt to new environments develop antibiotic resistance
•can interrupt functional genes
•can carry another gene
sit behind promoter and upregulate expression
of gene it carries
•variegation of color in corn
DNA Repair Mechanisms
Enzymes constantly scan DNA looking for errors.
Repair can occur during or after replication
Mutations which cripple the repair system
Xeroderma pimentosum
can not correct UV-induced damage to DNA
Mechanisms of Repair
A. DNA Polymerase corrects mistakes during replication
3’-5’ exonuclease activity
B. Mismatch Repair
wrong NT (non complementary base)
added during synthesis
corrected after replication
differentiates between methylated strand and unmethylated
C. Excision Repair Figure 14.27 and Figure 14.28
errors made from mutagens (UV rays, X rays)
NUCLEASE cuts out error
POLYMERASE replaces NT
LIGASE seals up
correction made post replication
Mutagenesis:Why Mutate?
Native proteins are not well suited for industrial application
Native proteins are not optimized for medicinal purposes
•Increase the efficiency of enzyme-catalyzed reactions
• Eliminate the need for cofactor in enzymatic reaction
•Change substrate binding site to increase specificity
•Change the thermal tolerance
•Change the pH stability
•Increase proteins resistance to proteases (purification)
Enzymes Used in Industrial Applications
amylase
lipase
papain
protease
beer production
cheese production
meat tenderizer
detergents
Protein
Engineering
Problems
Conditions of process inactivate the enzyme
high temperature
pH ranges
solvents
Specific Changes at the Protein Level
•Add Disulfide Bonds
•Changing Asparagine to Other amino Acids
Adding Disulfide Bonds
•Usually found in extracellular proteins, not intracellular
•Cross link between chains or in chains formed by
oxidation of cysteine residues
connective tissue
fibrin blood clots
• Why would we add disulfide bonds?
-Artificial addition may increase stability of protein
• As with all engineering AVIOD active site (enzyme)
XYLANASE
used to treat wood pulp in paper production
needs to function at high temp
Aspargine Changes
At high T, asparagine and glutamine deaminate
•ammonia is released
•amino acids convert to aspartic acid and glutamic acid
•Protein may refold
•LOSE ACTIVITY
Use Computer modeling to predict where mutation is preferred
Test stability/activity/stability of enzyme in vivo/in vitro
Reducing Free Sulfhydryl
Residues
Cysteine residues may cause dimerization
through intermolecular disulfide bonding
Convert Cys to another amino acid
reduce dimerization
maintain activity of enzyme
Enzyme Activity and Specificity
•Increase enzymatic activity by increasing affinity for enzyme
•change sequences in substrate binding site
•Change substrate of enzyme
Other Examples
Eliminate need for cofactors
Serine protease are used in laundry detergents
require calcium
modify enzymes ability to bind calcium
tPA tissue plasminogen activator
•dissolves blood clots
•would like to decrease clearance
•decrease non-specific bleeding
•increase specificity for fibrin in blood clot