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

Spontaneous? Induced?

Drosophila

Euphorbia

basal cell tumor

Mutation and natural selection

Point mutation • single base substitution, deletion, or addition

Point mutations Base pair substitutions transition = G to A transversion = ?

T to C

Missense mutation  Wrong amino acid encoded Sickle cell Hb

Subsititution, addition or deletion? TS or TV? Which codon position?

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

• Non-conservative mutation

Red blood cells

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 • Chemicals

Ethidium bromide

Mustard gas, industrial waste, PCBs, DDT

• Base analogues – Example: Bromouracil substitutes for thymine

• Radiation – sun (UV), radon, X-ray – Breaks, dimers… – High doses kill cells

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 to His+ colonies

1000X higher rate 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 p508

Will the mRNA message be in frame?

Effects of p508 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)