Transcript Examples of mutations and their phenotypes

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
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Site-directed mutagenesis-2
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• 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/ MoBio/Free/Ch9G.htm
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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
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More on IS and transposons
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• 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/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.
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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.
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Autosomal Dominant -1
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• 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.
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?
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Autosomal dominant -3
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• 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
Autosomal Recessive -1
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• 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.
Autosomal Recessive -2
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• 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.
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
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X-linked Diseases
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• 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.
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
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X-linked Diseases-2
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• 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
Trinucleotide repeats responsible for several
genetic diseases
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• 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
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–
–
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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
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Trinucleotide repeat diseases-2
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• 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.
Location of trinucleotide repeats
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