Mutations - Heritable changes in the base sequence of DNA
Silent mutations
Don't alter the phenotype
Silent because either:
1) Mutation occurs in non-coding or non-regulatory region
2) Mutation occurs in an intron (not at an intron/exon junction)
3) Mutation changes a codon such that it codes for the same amino acid
Base subtitutions
Can result in:
Missense mutation - changes an amino acid to another amino acid
Conservative substitution - changes to an amino acid with similar chemical properties
Radical replacement - changes to an amino acid with very different chemical properties
Nonsense mutation - changes an amino acid to a stop codon (UAA, UAG or UGA)
Deletions and Insertions
May result in frameshift mutation
Can revert frameshift by additional insertions/deletions
Inversions
Invert an entire region of the genome
Translocations
Move a part of the genome to a different location
Spontaneous vs. induced mutations
Mutagens - chemical or physical agents that can cause mutations
Nitrite - converts C to U
Intercalating agent - chemical that resembles a base pair of DNA and can insert between two base pairs
Acridine orange - causes DNA poly to insert an extra base during replication
UV radiation - causes the formation of thymine dimers
X-rays - cause multiple mutations and DNA rearrangements (insertions, translocations)
Transposons - "jumping genes" mobile pieces of DNA that can insert into a gene
Gene cassette - an engineered piece of DNA constructed with the express purpose of generating specific mutations. Inserted piece can be detected because of antibiotic resistance gene on the cassette.
Reversion - a second mutation that restores the original phenotype
True revertant - second mutation is at exact same site as the first
Second-site revertant - second mutation at a different site than the first mutation that cancels out the effects of the first mutation
Ames test
90% of all carcinogens are also mutagens
It's thought that cancer is caused by induction of certain mutations or by the accumulation of mutational damage in somatic cells. Some cancers may be caused by viruses.
Ames test - uses Salmonella typhimurium as test organism
2 His- strains (unable to synthesize histidine); both can revert to His+
Strain A reverts to His+ by base substitution
Strain B reverts to His+ by frameshift
Some compounds become mutagens only after interaction with certain enzymes in the human liver; that is, these enzymes convert the compound to a mutagen. These enzymes exist in the microsomal fraction of liver cells. So rat liver enzymes are added to the bacterial growth media in the Ames test.
Have 4 tubes:
1) Control: Rat liver enzymes + S. typhi. strain A (no chemical)
2) Rat liver enzymes + S. typhi. strain A + chemical being tested
3) Control: Rat liver enzymes + S. typhi. strain B (no chemical)
4) Rat liver enzymes + S. typhi. strain B + chemical being tested
Plate all on medium without histidine
Anything that grows is a revertant (either spontaneous [tubes 1 and 3] or induced [tubes 2 and 4]) and therefore was mutagenized. If get lots of revertants on plate 2, chemical is a mutagen that causes base substitutions. If get lots of revertants on plate 4, chemical is a mutagen that causes insertions or deletions. If get just a few revertants on all four plates, the chemical is not a mutagen. If get no revertents on any plates, the experiment was flawed (because you expect to get some revertants by spontaneous mutations that arose through mismatches incorporated during replication).
Positive Ames test doesn't mean that the compound is definitely a carcinogen. Positive proof that a specific compound is a carcinogen is determined from testing for tumor formation on laboratory animals.
DNA repair
Mismatch repair - corrects mistakes made in replication.
Mismatch repair enzymes follow behind replication fork; recognize bases that aren't hydrogen-bonded and introduces nicks in daughter strand. Segment of daughter strand is released, gap filled in by DNA pol I and final nick is sealed by ligase.
How does system distinguish between parent and daughter strands? Because of methylation. A residues of GATC sequences are methylated. Parent strand is fully methylated but daughter strand is not because hasn't been time (since replication) to methylate. So mismatch repair system excises nucleotides from undermethylated (daughter) strand.
Excision repair - repair T dimers
1) Incision - repair endonuclease recognizes distortion or kinks of T dimer. Cuts backbone near dimer
2) Displacement by pol I - pol I uses free 3'-OH of a single nick as primer for DNA synthesis. Synthesizes new strand while displacing strand containing dimer.
3) Excision - pol I releases displaced strand
4) Ligation - DNA ligase seals final nick
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Last updated: 9-April-98 / laa