Transcript Genetics Lecture 4 Mutation, Repair, Transposons (1)x

Geneticists  Mutants
Mutations are essential for:
Genetic analysis and gene mapping
Identifying and isolating disease genes
Understanding gene function
Discovering biochemical pathways
Evolution:
Most new mutations - deleterious
Some provide selective advantage
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Types of Mutations
Wild type
Mutant
Forward
Wild type
Reverse
(Backward)
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Types of Mutations
Point Mutations - Base-pair substitutions
transition
transversion
purpur; pyrpyr
purpyr; pyrpur
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Types of Mutations
Point Mutations - can change how codons are read
missense
nonsense
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Types of Mutations
Translation of a nonsense mutation
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Types of Mutations
Point Mutations - may not be obvious due to code redundancy
neutral
silent
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Types of Mutations
Point Mutations - can have polar effects
Frameshift:
insertion or
deletion
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Types of Mutations
Deletion - Null Mutant - ‘knock out’
Large segment or entire gene lost
No functional product possible
Reverse mutation impossible
unless gene replaced
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Suppressor Mutations
Second mutation cancels out effects of first
restores wild-type phenotype to mutants
Intragenic suppression
Both mutations in same gene
UGU (cys) - UGA (stop) - UGC (cys)
Intergenic suppression
Two different genes involved
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Intergenic Suppressor Mutations
Second mutation often in tRNA gene
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Intergenic Suppressor Mutations
Nonsense suppressor
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Intergenic Suppressor Mutations
Missense suppressor
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Classifying Mutations
Conditional - mutant phenotype expressed in certain conditions
Temperature sensitivity - tyrosinase (melanin production)
Useful for studying genes required for essential functions
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Classifying Mutations
Somatic - mutation occurs in body cells
affects only the individual
Germ line - mutation in gamete producing tissues
passed on to next generation
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Classifying Mutations
Spontaneous - random mistake
rate 1 in 104 to 109 mutations/cell/generation
Induced - caused by exposure to mutagen
mutagenesis
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Causes of Spontaneous Mutations
Tautomeric Shifts
enol form of G with T
imino form of A with C
imino form of C with A
enol form of T with G
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Causes of Spontaneous Mutations
Consequences of Tautomeric Shifts - transitions
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Causes of Spontaneous Mutations
DNA looping-out during replication (replication slippage)
Deletion
Insertion
Causes of Spontaneous Mutations
Replication slippage in trinucleotide repeat regions
Repeat expansion
Anticipation
Huntington disease
Fragile X syndrome
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Causes of Spontaneous Mutations
Deamination
C:G > U:A > T:A
A > Hypoxanthine:C
methylcytosine > T
C:G > T:A
Transitions
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Induced Mutations
Base analogs - 5-bromouracil
incorporated into DNA during synthesis
higher incidence of tautomeric shifts
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Induced Mutations
Base analogs - 5-bromouracil
Transitions
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Induced Mutations
Intercalating agents - misalignment mutagens
proflavin, acridine orange, ethidium bromide
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Induced Mutations
Intercalating agents - addition of nucleotide (base)
insertion
frameshift
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Induced Mutations
Intercalating agents - deletion of nucleotide (base)
deletion
frameshift
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Base Modifying Agents
Nitrous acid - oxidative deamination
Transitions
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Base Modifying Agents
Hydroxylamine (NH2OH)
Transitions
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Base Modifying Agents
Alkylating agents
Nitrogen mustard
CH3
Cl-CH2-CH2-N-CH2-CH2-Cl
Ethylmethanesulfonate
Nitrosoguanidine
CH3-Ch2-O-SO2-CH3
HN=C-NH-NO2
O=N-N-CH3
Transfer methyl or ethyl group to bases
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Base Modifying Agents
Alkylating agents
Methylmethane sulfonate
Transitions, Mispairing, Crosslinking and Breakage
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Mutagenic Effects of Radiation
Nonionizing radiation - Ultraviolet light (UV) - 260 nm
Absorbed by bases - pyrimidine hydrates, pyrimidine dimers
Mispairing,
Lethal if not
repaired
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Mutagenic Effects of Radiation
Ionizing radiation - Xrays, Radioactive Isotopes, Neutrons
(Radon gas, Radium)
High energy - penetrates tissues, displaces electrons
creates positively charged free radicals
Base changes, breaks in backbone, crosslinking
Results of exposure:
Base substitutions, Deletions, Duplications,
Inversions, Translocations, Chromosome breakage
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Self-Induced Mutagenesis
Radium - glows in the dark - watches, clothing
mouth cancer, build-up in bones, anemia
Nuclear energy - Chernobyl - 200x increase mutations in voles
X-rays
- physicians - bone cancer
shoe stores -
Tanning salons -
UVA/UVB both dangerous
Cigarette smoking - lung, pancreas, bladder, esophageal, etc.
Radon gas -
lung disease, cancer
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Identifying Mutagens
The Ames Test
liver extract
mimics metabolism
reverse mutations
induced
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Detecting Mutations
Visible - direct observation
Nutritional - auxotrophs
replica plating
Resistance - selective media
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Repair of DNA Damage
Spontaneous damage to DNA ~ 1 change/ 109 bp/min
10,000 mutations per cell every 24 hr
If not repaired, cells and individuals would die rapidly
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Light Repair - Photoreactivation
Direct repair of UV-induced pyrimidine dimers
Photolyase (phr) - activated by visible light
Error free repair
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prokaryotes, simple eukaryotes
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Repair of Alkylation Damage
O6-methylguanine methyltransferase (ada) - E. coli
removes methyl group restoring guanine
Similar mechanism for repair of alkylated thymine
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Base Excision Repair
Glycosylase recognizes and removes damaged base
by cleaving bond between base and sugar
Other enzymes remove the sugar leaving gap in DNA
DNA polymerase and DNA ligase repair gap
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Nucleotide Excision Repair (NER)
NER - Dark Repair - Repairs any damage that distorts DNA helix
E. coli
UvrA (uvrA), UvrB (uvrB), UvrC (uvrC), UvrD (uvrD)
UvrA and B recognize damage
UvrC and B cuts backbones on both sides of lesion
UvrD unwinds and releases region between cuts
DNA pol I and DNA ligase fill gap
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Nucleotide Excision Repair (NER)
E. coli
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Nucleotide Excision Repair (NER)
Mammalian systems - products of ~ 12 genes involved
Deficiency in repair - Xeroderma pigmentosum
light sensitivity
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Methyl-Directed Mismatch Repair
Recognizes mismatches
in newly synthesized DNA
E. Coli mutS, mutL, mutH
Exonuclease creates gap
DNA pol III and ligase
repair gap
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Methyl-Directed Mismatch Repair
Humans - hMSH2, hMLH1, hPMS1, hPMS2 involved
Mutations in any of these genes - HNPCC
Hereditary Nonpolyposis Colon Cancer
Autosomal Dominant - Predisposition to cancer
Heterozygous cell suffers mutation in good allele
No repair capability remains
Mutations begin to accumulate rapidly
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Double-Strand Break Repair
Homologous and Non-homologous recombination repair
Defects -
familial breast and ovarian cancer
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Recombination Repair
Postreplication - Recombination repair - recA
DNA pol and ligase
Nucleotide excision repair
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Translesion DNA Repair - SOS Response
E. coli - lexA, recA
Too much damage for repair, RecA is activated
induces LexA self-destruction
no more repression of 17 genes for SOS repair
DNA polymerase for translesion replication
introduces errors into DNA
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Transposable Elements
Mechanism for Movement -
insert into nonhomologous regions of chromosomes
Transposase:
move DNA elements
Eukaryotes and Prokaryotes
Reverse Transcriptase:
RNA > DNA > RNA
Eukaryotyes
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Transposable Elements in Prokaryotes
Phage mu - integration disrupts genes
ge *mu* ne
R plasmids - antibiotic resistance genes move
accumulate on plasmids - Multiple Resistance
ampRES
tetRES
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Transposable Elements in Prokaryotes
Insertion Sequences (IS) - 768 bp (IS1) , 4-19 copies
terminal inverted repeats (IRs), transposase
transposition into genes inactivates them, alters expression
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Insertion Sequences in Prokaryotes
IS movement into a nonhomologous target site
staggered
cut
direct
repeats
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Transposons in Prokaryotes
Transposons (Tn)
IS elements on
Both sides
Carry genes
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Transposon Movement
One possible model
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Transposable Elements in Eukaryotes
Barbara McClintock - Moveable genes in Indian corn (Zea mays)
Discovery 1940s; Noble Prize 1983; Died 1993 at 90
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Transposable Elements in Corn
Moveable genes in corn - controlling elements
C - purple
c - colorless
Insertion of Ds (dissociation)
C > c - colorless
Loss of Ds, reversion
c > C - purple spot
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Transposable Elements in Corn
Molecular evidence explains moveable genes in corn
Ac - activator
transposase
Ds no transposase
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Transposable Elements in Corn
Ac transposition mechanism
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Ty Elements in Yeast
Delta - promoter and recognition sequences for transposases
Two ORFs - TyA and TyB - encode proteins for transposition (RT)
Retrotransposons - move by an mRNA intermediate
~ 35 copies per genome
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P Elements in Drosophila
polypeptides
Approximately 15% of Drosophila genome is mobile.
P elements are one example.
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Human Retrotransposons
SINEs and LINEs are retrotransposons (interspersed elements)
~ 20% human genome = LINEs
~ 3% human genome = one SINE, Alu
Full length LINEs move autonomously,
also enable SINE movement
Transposition of LINE into factor VIII gene
responsible for spontaneous hemophilia
SINE insertion into neurofibromatosis gene caused disease
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