1Site-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

2Site-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.

3Site directed mutagenesis-3

www.web-books.com/ MoBio/Free/Ch9G.htm

4Transposons• 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

5More 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

6Examples 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.

7Human 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.

8Autosomal 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.

9Autosomal 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?

10Autosomal 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

11Autosomal 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.

12Autosomal 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.

13Autosomal 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

14X-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.

15X-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

16X-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

17Trinucleotide 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.

18Trinucleotide 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

19Trinucleotide 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.

20Location of trinucleotide repeats