Ch 7 Mutation• Heritable change in DNA

• Random

• Source of genetic variation

• may be advantageous, deleterious, neutral

Mutation (+ sexual reproduction) + natural selection = evolution

FYI: Adaptation vs mutation

• Adaptation – not proven– environment induces specific adaptive change– Ex. E. coli + ampicillin – some cells become

resistant in response

• Mutation– mutation is random– E. coli DNA mutates randomly – statistically, one

will result in amp resistance, this cell grows resulting in species adaptation

Types of mutations• germ-line mutation

– in sperm or egg (meiosis), will be passed to offspring

• somatic mutation– body cells, not passed on to offspring

• spontaneous mutation– natural aspect of DNA replication

• induced– UV light, chemicals, X-ray etc.

Germ line or somatic?

cat Drosophila Euphorbia basal cell tumor

Spontaneous?Induced?

Mutation and natural selection

Point mutation

• single base substitution, deletion, or addition

Point mutations

Base pair substitutionstransition = G to A T to C

transversion = ?

Missense mutation Wrong amino acid encoded

Sickle cell Hb

Subsititution, addition or deletion?

TS or TV?

Which codon position?

Red blood cells•Glu hydrophilic•Val hydrophobic (reduces solubility of the molecule)

•Non-conservative mutation

Conservative mutation

Example: Val to Leu both hydrophobic

Nonsense mutation– Premature stop codon – Codon AAA UAA

lys stop

5' - ATG ACT CAC CGA GCG CGA AGC TGA - 3‘

3' - TAC TGA GTG GCT CGC GCT TCG ACT

5' mRNA5' - AUG ACU CAC CGA GCG CGA AGC UGA - 3‘

Protein: Met Thr His Arg Ala Arg Ser Stop

5' - ATG ACT CAC TGA GCG CGA AGC TGA - 3‘

3' - TAC TGA GTG ACT CGC GCT TCG ACT - 5'

mRNA: 5' - AUG ACU CAC UGA GCG CGU AGC UGA - 3'

Protein: Met Thr His Stop

Neutral mutation

- mutation does not change amino acid folding

- codon AAA AGA

lys arg (both basic aa)

Silent mutation– No amino acid change, usually 3rd position TS

codon GCA GCG

arg arg

Frameshift mutation – Shifts reading frame -->

scrambled message

– May lead to a premature stop codon

The sun was hot but the man did not get his hat.

Transition or transversion and which codon position?

TATACCTAT

TATACCCTA

Other mutations

• Larger insertions, trinucleotide repeats, deletions, duplications, translocation, inversions

Trinucleotide repeat pg. 475 Fragile X Syndrome

• Xq27.3 FMR-1 gene

• X-linked dominant

• 2nd leading cause of mental retardation

(1/4000 males, 1/6000 females)

(Reprinted from Medical Genetics, 2nd ed., Jorde LB, et al, ©2000, with permission from Elsevier Science.)

• CGG repeat in promoter region– Normal = 6-54 repeats– Normal carriers = 55 – 200 repeats

(premutation expansion of repeat) – Fragile X syndrome = 200 – 1300 repeats

triplet repeat amplification

• FMR-1 FMRP (mRNA binding protein)

• FMRP regulates translation of mRNA

• Affects synaptic plasticity – signaling between neurons for learning and memory

• >200 repeats and FMR1 gene silenced retardation

• Caused by slipping of DNA polymerase during replication

Trinucleotide repeats

Mutation rate

Human estimate is ~1 x 10-6 spontaneous mutations/gene /generation

Mutation rates per gene differ.

May be hot spots

Causes of mutations

1. Spontaneous mistakes by DNA polymerase = replication errors

Most repaired by DNA proofreading and repair systems

2. Induced mutations

• ChemicalsMustard gas, industrial waste, PCBs, DDT

• Base analogues– Example: Bromouracil substitutes for thymine

• Radiation – sun (UV), radon, X-ray– Breaks, dimers…

– High doses kill cells

Ethidium bromide

The Ames test

• Indirect assay for carcinogens

• Plate 108 auxotrophic bacteria (his-)

• Add filter disc with chemical

• Look for revertants to his+

• = mutations

• Add rat liver extract to look at metabolism

Spontaneous reversion rate 1000X higher rateto His+ colonies with mutagen

Repair of mutations

1. Direct reversal

Example: Mismatch repair– 3’ 5’ proofreading by DNA polymerase

Also, UV repair (bacteria) and alkylation repair (prokaryotes)

2. Excision repair

Ex. base excision repair- single strand incision at both sides of error; - Excise mistake - DNA synthesis to replace the gap- Ligation

- Also UV repair prokaryotes, methyl mismatch repair, SOS response

Defective excision repair: Xeroderma pigmentosum (1/250,000) 9q34.1

• Autosomal recessive

• Extreme sun sensitivity– High risk of skin cancer (1000X) average by

age 8– Metastatic melanoma

• Corneal damage

Knockout mice for excision repair (XPA gene)

• XPA gene knocked out• UV sensitive and develop tumors• Study cancer, drugs

Small deletion example: Cystic fibrosis (1989)

• CFTR gene (chromosome 7)

• Encodes a transmembrane chloride channel active in cells that line the lungs

Cystic fibrosis recessive mutation 508

Will the mRNA message be in frame?

Effects of 508 CFTR mutation

• The 3 base deletion results in a channel protein that does not function correctly

• Thick mucus in the lungs, infections, lung stress

Phenotypic effects of mutation: Loss of function

• Drosophila white eye gene

• Wildtype = red eyes

Gain of function mutation

• Antennapedia (Drosophila)– legs on head (ectopic) in place of

antennae– Legs are normally expressed in

second thoracic segment

Knockout mice for excision repair (XPA gene)

• Genetically engineered to not express XPA gene• Mice are UV sensitive and develop tumors• Use mice for DNA repair/cancer study

Defective DNA mismatch repair: Hereditary nonpolyposis colon cancer

• Inherited predisposition to colon cancer

• ~4 people in family have had colorectal cancer, multiple generations, before age 50

Small deletion example: Cystic fibrosis (1989)