Download Mutations

Gene transfer
• Ways that bacteria can acquire new genetic info
– Transformation
• Taking up of “naked DNA” from solution
– Transduction
• Transfer of DNA one to cell to another by a virus
– Conjugation
• “Mating”: transfer of DNA from one bacterium to
another by direct contact.
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Transformation
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• Uptake of “naked” DNA
from medium.
• Classic experiment from
Genetics history, 1920’s.
Virulent cells have genes
for making capsule
which assists in
infection. Mutant cells
lack capsule, are
harmless. Griffith combined heat killed, virulent cells with
live, harmless mutants. The living cells took up
the DNA from solution, changed into capsuleproducing, virulent bacteria.
Transformation details
DNA must be homologous, so transformation
only occurs between a few, close relatives.
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Transduction
• Transfer of DNA via a virus.
More common,
but still requires
close relative.
Conjugation: bacterial sex
• If sex is the exchange of genetic
material, this is as close as bacteria
get. Conjugation is widespread and
does NOT require bacteria to be
closely related.
• Bacteria attach by means of a sex
pilus, hold each other close, and DNA
is transferred.
• Plasmids such as F plasmids and R
plasmids can be exchanged, leading
to antibiotic-resistant bacteria.
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“feminist’s nightmare”
• F+ cells (donor) are considered “male”, recipient cells (F-)
are considered female.
• F+ cells transfer a copy of the F plasmid to the recipient,
therefore making it “male” also.
http://fig.cox.miami.edu/Faculty/Dana/ffactor.jpg
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Conjugation can result in transfer of
chromosomal genes
• F plasmid can insert and excise, bringing with it a piece of
chromosomal DNA = F’
• F plasmid stays inserted, directs copying and exchange of
chromosomal DNA, but not F plasmid: Hfr cell.
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Mutations
A mutation is an inheritable change in DNA.
This means an alteration in a basepair or
in the order of the basepairs.
Mutations may affect a single basepair,
(point mutation) where they may
change the sequence in an RNA or
protein, or not (silent mutation).
During protein synthesis, bases are read
3 at a time (codon); when the first base
is read, the “reading frame” is established.
If the frame is altered at some point the bases will NOT be in
the right places, and the information will be garbled.
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Mutagens
• Radiation
– Ionizing radiation chemically alters
DNA, breaks one or two DNA strands
• Leads to replication failure, death
– Ultraviolet radiation causes thymine
dimers to form
• Kink in DNA chain also leads to
replication failure unless repaired.
http://www.bio.cmu.edu/Courses/03441/TermPapers/96TermPapers/spontaneous/dimer-2.jpg
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Mutagens-2
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• Chemical changes
– Many mutagens are chemicals that change one base to
another, cause mispairing.
– Others are base analogs, used instead of real base, cause
mispairing or disrupt replication.
• Frameshifts
– Flat molecules (ethidium bromide, acridine orange)
intercalate into DNA (slip between flat bases).
• When replication occurs, extra base added, disrupts
reading frame, scrambles codons.
Non-mutagen cause of mutations
(causes background level)
• Copying errors
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– Fairly rare; enzyme messes up and doesn’t catch mistake
– DNA polymerase has a 3’ to 5’ exonuclease activity that
backspaces, deletes mistakes.
• Bases themselves undergo spontaneous change to
wrong base. Repair sometimes doesn’t happen.
– Cytosine to uracil change is detected, removed.
– Missing purines are detected, repaired.
DNA damaged often repaired
• The mutation rate is variable
– Typical 1^106 per gene per generation
– Rare, so DNA damage must be frequently repaired.
• Repair of thymine dimers
– Excision repair: bad spot of DNA cut out, replaced
– Light repair: a photon of blue light + enzyme undoes it.
• Mismatch repair: if base pairs aren’t a pair
– Stretch of newer DNA is cut out, replaced.
• SOS repair: too much damage!!
– Base pair “fidelity” is relaxed to save time. More
mutations produced, but cells live.
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Using mutations-1
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• The Ames test
– Developed by biochemist Bruce Ames to determine
whether a substance is a mutagen
• Nearly all carcinogens are mutagens; first level screen.
– His- mutant of Salmonella combined with chemical
• Plated onto medium without histidine, won’t grow
• If chemical is a mutagen, revertants will appear at high
frequency, no longer need histidine in medium.
• Test often done with liver extract; enzymes mimic
human body where metabolite may be mutagen.
Using mutations-2
• How to get a mutant like the his- mutant?
– Treat with a mutagen (UV light, chemical, etc.)
– Spread survivors onto rich nutrient medium, get
colonies.
• Many bacterial mutants that are studied have nutritional
defects.
– Wild type (normal) is called a prototroph
– Mutant that can’t make a nutrient is called an
auxotroph.
• This example: looking for a serine auxotroph.
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Replica plating
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First, expose cells to mutagen, then spread onto defined
medium containing many amino acids and other nutrients.
Replica plate onto medium with same composition except NO
SERINE.
Result of replica plating
• Look: where did a colony NOT grow on this plate?
• Go back to original plate and grow colony.
www.sp.uconn.edu/.../ lectures/genetics1.html
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Transposon mutagenesis and positive
selection of mutants
• Transposons
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– Piece of DNA that can copy itself and insert at random
into the bacterial chromosome.
– Contains gene for transposase, DNA sequences needed
for insertion, and an antibiotic resistance gene.
• Transposons are useful for making mutants
– Insertion is random, so there is the possibility of mutating
the gene you are looking for.
– Antibiotic resistance provides a “selectable marker”
Transposon mutagenesis-2
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• Use a virus or conjugating donor bacterium to
introduce Tn5 into recipient.
• Once DNA with Tn5 is in cell, transposon jumps
once into recipient’s DNA, causing a different
mutation in each cell.
• Plate recipient cells onto kanamycin-containing
agar; only cells with transposon mutations survive.
– This is positive selection
• Mutations can now be mapped, cloned, or whatever
else you wish to do.
Tools and Techniques of genetic engineering
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• Reverse transcriptase: an enzyme that makes DNA
from an RNA copy
– Naturally produced by retroviruses
– For cloning eukaryotic DNA into bacteria
– Eukaryotic DNA has introns that prokaryotic cells don’t
have and can’t deal with
• If you make DNA directly from mRNA, the introns
have already been removed
Tools and Techniques-2
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• Restriction enzymes (restriction
endonucleases)
– Made by bacteria to destroy DNA
from viruses
– Recognize 6 (or 4) base DNA
sequences and cut them
• Palindromes like GAATTC, cut
staggered, leaving sticky ends.
• Sticky ends are the key: allow
DNA from ANY source to be cut,
combined, and stitched together.
http://employees.csbsju.edu/hjakubowski/classes/ch
331/dna/restriction.gif
Tools and Techniques-3
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• Cloning
– To work with a piece of DNA, you need enough
molecules of it (identical molecules)
– Vectors
• A way to move a piece of DNA into a cell to make
multiple copies of it (clone)
• Usually a plasmid or something similar; cut with
restriction enzyme, paste in DNA, use transformation
or transduction to get it into the bacterium.
• Plasmid will copy itself, so will bacterium.
Tools and Techniques-4
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• Cloning-2
– PCR: polymerase chain reaction
– If you know the sequence on either side of the DNA, you
can use PCR.
– PCR “amplifies” DNA, so you can get multiple copies of
identical DNA molecules this way.
– Requires dNTPs, heat stable polymerase (Taq), primers
complementary to piece of DNA, and your DNA.
– PCR incredibly useful for wide variety of applications.
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PCR
http://members.aol.com/BearFl
ag45/Biology1A/LectureNotes
/LNPics/Recomb/pcr.gif