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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 Wild type Forward Reverse

(Backward)

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Types of Mutations

Point Mutations - Base-pair substitutions

transition

transversion

purpur; pyrpyr

purpyr; pyrpur

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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 impossibleunless gene replaced

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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

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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

Causes of Spontaneous Mutations

DNA looping-out during replication (replication slippage)

Deletion

Insertion

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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 > TC: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)

insertionframeshift

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Induced Mutations

Intercalating agents - deletion of nucleotide (base)

deletionframeshift

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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

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

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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 notrepaired

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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 extractmimics metabolism

reverse mutationsinduced

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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 - 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 ligaserepair 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 remainsMutations 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 replicationintroduces 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

R plasmids - antibiotic resistance genes move

accumulate on plasmids - Multiple Resistance

ge *mu* ne

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

Approximately 15% of Drosophila genome is mobile.

P elements are one example.

polypeptides

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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 generesponsible for spontaneous

hemophilia

SINE insertion into neurofibromatosis gene caused disease