Site-directed mutagenesis

1. 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

2. Site-directed mutagenesis-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.

3. Site directed mutagenesis-3 www.web-books.com/ MoBio/Free/Ch9G.htm

4. 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

5. More on IS and transposons • 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/MicrobialGenetics/topics/transposons/non-repl-tpn.html

6. 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, non-functional protein to be produced. • Mutations are only source of new alleles, but also the cause of genetic disease.

7. 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.

8. Autosomal Dominant -1 • 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 4p • 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.

9. Autosomal dominant -2 • Marfan syndrome • Disease among group: connective tissue disorders • Caused by deficiency of fibrillin, required for proper connective tissue production. • Map location: 15q21.1 • 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?

10. Autosomal dominant -3 • Achondroplasia • Gene located 4p16.3 • 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

11. Autosomal Recessive -1 • Cystic fibrosis • Caucasian populations: heterozygotes 1/25, occurrence in newborns (north. Europ. 1/2000) • CF gene maps to 7q31.2 • 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.

12. Autosomal Recessive -2 • 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, O2 deprivation of tissues. • Selection is positive because protection against malaria. 11p15.5 is map location.

13. 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 • 20q13.11 • Tay-Sachs disease • Map: 15q23-24; 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

14. X-linked Diseases • Fragile X syndrome • 2nd Leading cause of inherited mental retardation after Down Syndrome. 1/2500 children • Site Xq27-28 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.

15. 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 Xp21.2

16. X-linked Diseases-2 • Hemophilia-A • Failure of blood to clot, lack of factor VIII. • The incidence of hemophilia is about 1:7,500 live male births and 1:25,000,000 live female births. • maps to Xq28. 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. • Xq28

17. Trinucleotide repeats responsible for several genetic diseases • 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.

18. 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

19. Trinucleotide repeat diseases-2 • Fragile X syndrome (Martin-Bell) • X linked trait, but more common in females because it is dominant (1 in 8000 vs. 1 in 4000) • 2nd 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.

20. Location of trinucleotide repeats