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? catDrosophila Euphorbiabasal cell tumor Spontaneous? Induced?

Mutation and natural selection

Point mutation single base substitution, deletion, or addition

Point mutations Base pair substitutions transition = 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 3 rd 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 2 nd 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 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 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 Ethidium bromide

The Ames test Indirect assay for carcinogens Plate 10 8 auxotrophic bacteria (his-) Add filter disc with chemical Look for revertants to his+ = mutations Add rat liver extract to look at metabolism

Spontaneous reversion rate1000X higher rate to His+ colonieswith 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)