Site-directed mutagenesis Mutation is a random process

Site-directed mutagenesis • Mutation is a random process – The rate of mutation can be increased with radiation or mutagenic chemicals – Individuals mutant at certain loci can be selected for or screened for, but the exact location and nature of the mutation is unknown. • Site-directed allows for a particular mutation to be created in a particular location in the DNA – Requires knowledge of the DNA sequence – Requires ability to synthesize oligonucleotides 1

Site-directed mutagenesis-2 2 • Procedure – Oligonucleotide with desired mutation is chemically synthesized – Ss DNA is obtained from cell • Inserted in a plasmid – The two are hybridized then the oligonucleotide is extended to make ds DNA. – DNA synthesis (semi-conservative) produces one normal DNA molecule, one mutant molecule, leading to one normal cell, one mutant cell.

Site directed mutagenesis-3 www. web-books. com/ Mo. Bio/Free/Ch 9 G. htm 3

Transposons • DNA can “jump” from one location to another – Three kinds of examples • Insertion sequences (simple) (IS) • Transposons (made of IS plus other genes) • Certain viruses like Mu that insert themselves • Transposition can be replicative or not – Non-replicative: DNA “element” physically moves to new location within the DNA – Replicative: DNA element is copied to another location. • Transposition occurs in both pro- & eukaryotes 4

More on IS and transposons 5 • IS: have inverted terminal repeats and code for a transposase that moves the IS. • Tranposons have IS at each end and unrelated genes in the middle. A site that discusses transposons and replicative vs. non -replicative transposition: http: //www. sci. sdsu. edu/~smaloy/Microbial. Genetics/topic s/transposons/non-repl-tpn. html

Examples of mutations and their phenotypes • ABO blood groups – Enzymes involved are glycosyltransferases, add carbohydrates to lipids in membranes – Type A and Type B alleles differ by 4 nucleotides – Type O (only H substance produced) • Gene contains early frameshift, causes short, nonfunctional protein to be produced. • Mutations are only source of new alleles, but also the cause of genetic disease. 6

Human Genetic Diseases • Presented because of general interest in human, medical genetics. • Note the following information: – Name of disease; frequency in population; gender, race, or ethnicity of most affected individuals, disease symptoms, chromosomal location, cause of disease if known. 7

Autosomal Dominant -1 8 • Huntington’s Disease – 1/10, 000 in people of European descent. – Progressive deterioration of nervous system • Jerky spasmodic movements, then loss of control • Require total care in last years of life – Onset age 30 -50, duration 10 -15 yr ending in death – Gene linked to RFLP near end of 4 p • Trinucleotide repeat CAG; 11 -34 copies normal, 42 to >100 in disease – Function of protein, Huntingtin, elusive. Positively affects brain. Would be 3144 amino acids long.

Autosomal dominant -2 • Marfan syndrome – Disease among group: connective tissue disorders – Caused by deficiency of fibrillin, required for proper connective tissue production. – Map location: 15 q 21. 1 – Incidence: 1/10, 000. 25% are spontaneous mut. – Syndrome: skeleton, lens of eye, CV system • Tall with long thin fingers and arms • Aorta susceptible to rupture • Flo Hyman, Abe Lincoln? 9

Autosomal dominant -3 10 • Achondroplasia – Gene located 4 p 16. 3 – Frequency is 1/50, 000, mostly spontaneous mut. – Protein affected is fibroblast growth factor receptor • Interferes with cartilage production • Head and trunk normal, long bones don’t grow – Not cured by human growth hormone – Cure? Elizaroff technique: break and extend bones, force new growth. – Herve Villachez of original Fantasy Island TV show

Autosomal Recessive -1 11 • Cystic fibrosis – Caucasian populations: heterozygotes 1/25, occurrence in newborns (north. Europ. 1/2000) – CF gene maps to 7 q 31. 2 – Many possible mutations in area, 20 very common, one (70% of all) most common: 3 base deletion. – Protein is an ion channel, 1480 aa, regulates flow of salt through epithelial cell membranes. • Failure: water retained, thick mucus, salty sweat, chronic infections and malfunction. – Life expectancy much improved; quality still poor.

Autosomal Recessive -2 12 • Sickle cell anemia – In people of Mediterranean or African origin • In US, 1/500 Afr-Amer afflicted, 1/12 heterozyg. – Single point mutation in hemoglobin gene • Causes protein to become sticky, especially w/o Oxygen bound to it. Episodes when winded. – Aggregation of Hem. makes clumps, RBC become sickle shaped; clog capillaries, break. • Anemia, pain, O 2 deprivation of tissues. – Selection is positive because protection against malaria. 11 p 15. 5 is map location.

Autosomal Recessive -3 • Adenosine deaminase deficiency severe combined immunodeficiency disease (ADD) – Catabolism of adenosine to uric acid is blocked • Deoxyadenosine accumulates, kills helper T cells – 20 q 13. 11 • Tay-Sachs disease – Map: 15 q 23 -24; no synthesis of hexosaminidase A • Excess fatty acids accumulate, nerve cells killed – Various forms and stages, classic is < 6 mo infant – Most common in Ashkenazaic Jews, 1/3600 in US 13

X-linked Diseases 14 • Fragile X syndrome – 2 nd Leading cause of inherited mental retardation after Down Syndrome. 1/2500 children – Site Xq 27 -28 which breaks in cell culture when starved for nucleotides. – 3 things to learn from this • Example of sex-linked (X-linked) inheritance – But dominant instead of recessive. • Another trinucleotide repeat disease • Phenomenon called imprinting – Transmitting males not affected, but daughters are.

X-linked Diseases-2 • Duchenne Muscular Dystrophy – Loss of muscle function starting w/ voluntary muscles, then involuntary – Symptoms begin early, life expectancy <30 yrs • Affects boys, 1/3500 – Gene for large protein “dystrophin” is mutated • Dystrophin gene contains any of various deletions or additions resulting in frameshift mutations; • Protein is defective; fails to anchor cytoskeleton to membrane proteins; cells die • Weakens muscle fibers. – Gene location Xp 21. 2 15

X-linked Diseases-2 16 • Hemophilia-A – Failure of blood to clot, lack of factor VIII. – The incidence of hemophilia is about 1: 7, 500 live male births and 1: 25, 000 live female births. – maps to Xq 28. Various mutations occur in the gene • Red-green colorblindness – Not a “disease”, a condition – Failure to produce protein-pigment light receptors needed to perceive colors. – Xq 28

Trinucleotide repeats responsible for several genetic diseases 17 • In these genes, the repeat is normal – Excessive numbers of repeat causes disease – The higher the # of repeats, the earlier and more severe • Genetic anticipation – When the number of repeats is high, offspring inherit increasingly higher numbers of repeats, worse disease • Causes of diseases are unknown – The repeats can occur in various locations; not clear why large numbers of repeats cause disease. See below.

Trinucleotide repeat diseases-1 • Huntington Disease – Complete loss of muscle control with age; dominant – CAG repeat in gene for “huntingtin” – 10 -35 repeats normal; up to 120 in disease – Repeat located in coding portion: calls for glutamine • Myotonic dystrophy – Weakness of muscles and other affects – Dominant gene on chromosome #19 – Repeat is in 3’ untranslated region of gene for protein kinase: CTG – 5 -37 copies is normal; up to 1500 copies diseased 18

Trinucleotide repeat diseases-2 19 • Fragile X syndrome (Martin-Bell) – X linked trait, but more common in females because it is dominant (1 in 8000 vs. 1 in 4000) – 2 nd leading cause of mental retardation – Thin section of X chromosome breaks in cell culture when cells starved for certain nutrients (folic acid) – At thin section, FMR-1 gene has CGG repeat • Actually upstream from coding sequence – 6 -54 repeats normal; 55 -230 repeats normal BUT passes on an increased # to offspring; above 230, retarded.

Location of trinucleotide repeats 20