Upload
erika-hart
View
216
Download
0
Tags:
Embed Size (px)
Citation preview
1
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
2
Types of Mutations
Wild type Mutant Wild type Forward Reverse
(Backward)
3
Types of Mutations
Point Mutations - Base-pair substitutions
transition
transversion
purpur; pyrpyr
purpyr; pyrpur
4
Types of Mutations
Point Mutations - can change how codons are read
missense
nonsense
5
Types of Mutations
Translation of a nonsense mutation
6
Types of Mutations
Point Mutations - may not be obvious due to code redundancy
neutral
silent
7
Types of Mutations
Point Mutations - can have polar effects
Frameshift:
insertion or deletion
8
Types of Mutations
Deletion - Null Mutant - ‘knock out’
Large segment or entire gene lost
No functional product possible
Reverse mutation impossibleunless gene replaced
9
Suppressor Mutations
Second mutation cancels out effects of firstrestores wild-type phenotype to mutants
Intragenic suppressionBoth mutations in same gene
UGU (cys) - UGA (stop) - UGC (cys)
Intergenic suppressionTwo different genes involved
10
Intergenic Suppressor Mutations
Second mutation often in tRNA gene
11
Intergenic Suppressor Mutations
Nonsense suppressor
12
Intergenic Suppressor Mutations
Missense suppressor
13
Classifying Mutations
Conditional - mutant phenotype expressed in certain conditions
Temperature sensitivity - tyrosinase (melanin production)
Useful for studying genes required for essential functions
14
Classifying Mutations
Somatic - mutation occurs in body cells
affects only the individual
Germ line - mutation in gamete producing tissues
passed on to next generation
15
Classifying Mutations
Spontaneous - random mistake
rate 1 in 104 to 109 mutations/cell/generation
Induced - caused by exposure to mutagen
mutagenesis
16
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
17
Causes of Spontaneous Mutations
Consequences of Tautomeric Shifts - transitions
Causes of Spontaneous Mutations
DNA looping-out during replication (replication slippage)
Deletion
Insertion
19
Causes of Spontaneous Mutations
Replication slippage in trinucleotide repeat regions
Repeat expansion
Anticipation
Huntington disease
Fragile X syndrome
20
Causes of Spontaneous Mutations
Deamination
C:G > U:A > T:A
A > Hypoxanthine:C
methylcytosine > TC:G > T:A
Transitions
21
Induced Mutations
Base analogs - 5-bromouracil
incorporated into DNA during synthesis
higher incidence of tautomeric shifts
22
Induced Mutations
Base analogs - 5-bromouracil
Transitions
23
Induced Mutations
Intercalating agents - misalignment mutagens
proflavin, acridine orange, ethidium bromide
24
Induced Mutations
Intercalating agents - addition of nucleotide (base)
insertionframeshift
25
Induced Mutations
Intercalating agents - deletion of nucleotide (base)
deletionframeshift
26
Base Modifying Agents
Nitrous acid - oxidative deamination Transitions
27
Base Modifying Agents
Hydroxylamine (NH2OH)
Transitions
28
Base Modifying Agents
Alkylating agents
CH3
Nitrogen mustard Cl-CH2-CH2-N-CH2-CH2-Cl
Ethylmethanesulfonate CH3-Ch2-O-SO2-CH3
Nitrosoguanidine HN=C-NH-NO2
O=N-N-CH3
Transfer methyl or ethyl group to bases
29
Base Modifying Agents
Alkylating agents
Methylmethane sulfonate
Transitions, Mispairing, Crosslinking and Breakage
30
Mutagenic Effects of Radiation
Nonionizing radiation - Ultraviolet light (UV) - 260 nm
Absorbed by bases - pyrimidine hydrates, pyrimidine
dimers
Mispairing,Lethal if notrepaired
31
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
32
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
33
Identifying Mutagens
The Ames Test
liver extractmimics metabolism
reverse mutationsinduced
34
Detecting Mutations
Visible - direct observation
Nutritional - auxotrophs
replica plating
Resistance - selective media
35
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
36
Light Repair - Photoreactivation
Direct repair of UV-induced pyrimidine dimers
Photolyase (phr) - activated by visible light
Error free repair - prokaryotes, simple eukaryotes
37
Repair of Alkylation Damage
O6-methylguanine methyltransferase (ada) - E. coli
removes methyl group restoring guanine
Similar mechanism for repair of alkylated thymine
38
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
39
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
40
Nucleotide Excision Repair (NER)
E. coli
41
Nucleotide Excision Repair (NER)
Mammalian systems - products of ~ 12 genes involved
Deficiency in repair - Xeroderma pigmentosum
light sensitivity
42
Methyl-Directed Mismatch Repair
Recognizes mismatches in newly synthesized DNA
E. Coli - mutS, mutL, mutH
Exonuclease creates gap
DNA pol III and ligaserepair gap
43
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 remainsMutations begin to accumulate rapidly
44
Double-Strand Break Repair
Homologous and Non-homologous recombination repair
Defects - familial breast and ovarian cancer
45
Recombination Repair
Postreplication - Recombination repair - recA
DNA pol and ligase
Nucleotide excision repair
46
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 replicationintroduces errors into DNA
47
Transposable Elements
Mechanism for Movement -
insert into nonhomologous regions of chromosomes
Transposase: move DNA elements
Eukaryotes and Prokaryotes
Reverse Transcriptase:
RNA > DNA > RNA
Eukaryotyes
48
Transposable Elements in Prokaryotes
Phage mu - integration disrupts genes
R plasmids - antibiotic resistance genes move
accumulate on plasmids - Multiple Resistance
ge *mu* ne
ampRES
tetRES
49
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
50
Insertion Sequences in Prokaryotes
IS movement into a nonhomologous target site
staggered cut
direct repeats
51
Transposons in Prokaryotes
Transposons (Tn)
IS elements on Both sides
Carry genes
52
Transposon Movement
One possible model
53
Transposable Elements in Eukaryotes
Barbara McClintock - Moveable genes in Indian corn (Zea mays)
Discovery 1940s; Noble Prize 1983; Died 1993 at 90
54
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
55
Transposable Elements in Corn
Molecular evidence explains moveable genes in corn
Ac - activator transposase
Ds - no transposase
56
Transposable Elements in Corn
Ac transposition mechanism
57
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
58
P Elements in Drosophila
Approximately 15% of Drosophila genome is mobile.
P elements are one example.
polypeptides
59
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 generesponsible for spontaneous
hemophilia
SINE insertion into neurofibromatosis gene caused disease