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

• Huntingtons 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 gt100 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 dont 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 lt 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 lt30 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 17,500
  live male births and 125,000,000 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