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© 2006 Jones and Bartlett Publishers
Chapter 12 Opening photo. Peacock [© Photos.com]
altered information
heritable change in the DNA (?)
Mutations:
Classification
spontaneous vs. induced
randomunpredictablerates
radiationchemical
mutagen
Ames test
Classification
somatic vs. germ line
body cells“mosaics”most cancers
gametespassed on
Classification
conditional vs. unconditional
on permissiveconditions
off restrictiveconditions
e.g.,temperature sensitive mutants
expressed all the time
© 2006 Jones and Bartlett Publishers
Fig. 12.1. A Siamese cat showing the characteristic pattern of pigment deposition [Courtesy of Jen Vertullo]
Cats:enzyme for melanin deposition is temperature sensitive
Off at normal body temperatureOn at cooler temperatures
(face, paws, tail)
Classification
based on their affect on gene
loss of function(knockout)hypomorhphic (leaky)hypermorphicgain of function (also ectopic exp.)
recessive
dominant
belowabove
Classification
based on molecular changes
base substitution
5’-GAG-3’3’-CTC-5’
5’-GAG-3’3’-CAC-5’
5’-GAG-3’
3’-CAC-5’
3’-CTC-5’
5’-GTG-3’
unmutated
mutant
Classification
based on molecular changes
base substitution
transition: pyrimidine purinepyrimidine purine
transversion: pyrimidine to purinepurine to pyrimidine
T C, C T: A G, G A
How many?
more common
Classification
based on molecular changes
base insertions or deletions
(wait three slides)
base substitutions in coding region:
missense
silent
nonsense
frameshift
change of amino acid
doesn’t change amino acid
make a new stop codon
small insertion of deletion shifts reading frame
(pos
ition
)
Chapter 8 Gene Expression
missense
silent
nonsense
?
?
Table 1.1
© 2006 Jones and Bartlett Publishers
Fig. 8.23. Reading bases in an RNA molecule
codons are linear and non-overlapping
© 2006 Jones and Bartlett Publishers
Fig. 8.24. Change in an amino acid sequence of a protein caused by the addition of an extra base
frameshift mutationreading frame
insertion
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Fig. 8.25. Interpretation of the rll frameshift mutations
© 2006 Jones and Bartlett Publishers
Table 12.1. Major types of mutations and their distinguishing features
One of the “classic” mutations is
sickle cell anemia
single base substitution (missense)
amino acid #6 from glutamic acidto
valine
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Fig. 12.3. Base substitution mutation in sickle-cell anemia
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-hemoglobinA
-hemoglobinS
forms long needle like crystalsRBC’s become sickle shaped
normal protein foldingnormal RBC’s
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HbA
HbA
HbA
HbS
HbS
HbS
“normal”
sickle cell trait
sickle cell disease
some symptoms
often die young
dynamic mutations
X chromosomeinstability in region of CGG repeatreplication slippage
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Fig. 12.4. Pedigree showing transmission of the fragile-X syndrome. [After C. D. Laird. 1987. Genetics 117: 587]
12.4
mutations are random, but……they also happen at characteristic rates
(they do not arise in response to conditions)
agar plates with antibiotic
plate antibiotic-sensitive bacteria
some colonies grow (have resistance)
induced?or
random?
replica plating (Lederberg’s)
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Fig. 12.13. Replica plating
mutants grow in the same place,therefore the mutations occurred before they were plated
Selective techniques merely selectmutants that pre-exist in a population
development of resistance to:antibiotics (pesticides, etc.,)
DDT resistant insectsMRSATB in Russian prisons
Mutations
random
consistent
variable
can’t predict where they happen
they happen at a measurable frequency
rate varies from gene to geneand organism to organism
Mutational hotspots
places where mutations are more likely to occur
trinucleotide repeatsmethylated cytosine
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Fig. 12.15. Loss of the amino group in 5-methylcytosine and from normal cytosine
removed/repaired by
uracil glycosylase
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Table 12.3. Major agents of mutation and their mechanism of action
What are the physical causes of mutations?
Water
depurination
removal of base from purine nucleotide
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Fig. 12.16. Depurination
Water
depurination
removal of base from purine nucleotide
can be repaired
mutation rate (in air?):
3 depurinations109 purines
per minute
nitrous acid
can deaminate A C G
H U
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Fig. 12.17. Deamination of adenine results in hypoxanthine
thymine cytosine
nitrous acid
can deaminate A C G
H U
C
?
base analogs
can substitute for normal base
more likely to mis-pair than normal bases
5-bromouracil (Bu) similar to thymidine
keto-enol-
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Fig. 12.18. Mispairing mutagenesis by 5-bromouracil
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Fig. 12.18. Mispairing mutagenesis by 5-bromouracil
© 2006 Jones and Bartlett Publishers
Fig. 12.19. Two pathways for mutagenesis by 5-bromouracil
alkylating agents
EMS ethyl methanesulfonate
nitrogen mustard
Fig. 12.20. Chemical structures of two highly mutagenic alkylating agents
© 2006 Jones and Bartlett Publishers
Fig. 12.21. Mutagenesis of guanine by ethyl methanesulfonate (EMS)
mispairing
Intercalating agents
interfere with topoisomerase II (gyrase)
leaves nicks in DNA
leads to deletions or insertions
(EtBr)
http://en.wikipedia.org/wiki/Ethidium_bromide
UV light
causes formation of T-T dimers
distorts double helix
interferes with transcriptiontranslation
© 2006 Jones and Bartlett Publishers
Fig. 12.22. (A) Formation of a thymine dimer (B) distortion of the DNA helix caused by two thymines
Ionizing radiation
X-raysparticles/radiation from radioactive decay
Over a wide range of X-ray doses:
frequency of mutationproportional to
radiation dose
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Fig. 12.23. Relationship between the percentage of x-linked recessive lethals and x-ray dose in D. melanogaster
Three types of damage from ionizing radiation
single-strand breakage
double-strand breakagenucleotide alterationchromosome breaks
usually repaired
radiationtherapy
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Fig. 12.24. Annual exposure of human beings in the United States to various forms of ionizing radiation. [Source: National Research Council]
Chernobyl
1986
10X radiation of bombing of Hiroshimabut little damage was expected
1996 mutations rates were 2X(for some loci)
© 2006 Jones and Bartlett Publishers
Fig. 12.25. Mutation rates of five tandem repeats among people of Belarus who were exposed to radiation from Chernobyl and among unexposed British people. [Data from Y. E. Dubrova, et. al. 1996. Nature 380: 183.]
Fixing DNA damage
1 mutationbillion bp
per minute
every cell would have damage at 10,000 sites every 24 hours
12.5
Mendelian genetics (2)Modifications to MendelSex determination and sex-linkage (3)
Linkage and crossing overincluding two and three point test crosses (4)
and including lab materialQuantitative genetics (15)Mutation and DNA repair (12)Problem set #1Problem set #2
Exam 3 Next Friday 4/4
No class (tentatively) 4/18
© 2006 Jones and Bartlett Publishers
Table 12.6 Types of DNA damage and mechanisms of repair
DNA ligase
uracil glycosylase
mismatch repairAP endonucleaseenzymatic reversalexcision repair
postreplication repair
* *
mismatch repair
“last-chance” error correction for mistakes
mismatch repair
detect mismatch
cut one strand on either side of mistake
Which strand?
strand that is least methylated
mismatch repair
detect mismatch
cut one strand on either side of mistake
remove that strand in area of mismatch
fill in missing strand
mismatch repair in prokaryotes
protein from mutSprotein from mutL
recognize and bind to mismatches
protein from mutH makes a nick in bad strand
exonuclease
DNA PolDNA Ligase
removes strand past mistake
fills in new complimentseals the nick
© 2006 Jones and Bartlett Publishers
Fig. 12.27. Mismatch repair
mismatch repair in prokaryotes
protein from mutSprotein from mutL
recognize and bind to mismatches
protein from mutH makes a nick in bad strand
exonuclease
DNA PolDNA Ligase
removes strand past mistake
fills in new complimentseals the nick
mismatch repair in prokaryotes
mutSmutL
bacteria with defects in either of these have high rates of spontaneous mutations
four homologous genes in humans
mutations in any may lead to HNPCC
(human nonpolyposis colorectal cancer)
AP endonuclease filling in gaps
deaminationhydrolysis
(apyrimdinic site)(apurinic site)
C to UA,T to -OH
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Fig. 12.28. Action of AP endonuclease
UV damage (cross-links)
enzymes that reverse linkage
excision repair
endonuclease cut on either side of damage
DNA pol. displaces damaged segmentfills in new compliment
DNA ligase joins ends together
© 2006 Jones and Bartlett Publishers
Fig. 12.29. Mechanism of excision repair of damage to DNA
endonuclease
postreplication repair (PRR)
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Fig. 12.30. Postreplication repair
12.7 Testing for mutagens
The Ames test
histidine requiring (His-) mutants of Salmonella
test for reversion (to His+)
sensitive, quantitative
© 2006 Jones and Bartlett Publishers
Fig. 12.31. Linear dose–response relationships obtained with various chemical mutagens in the Ames test.. [Data from B. N. Ames. 1974. Science 204: 587–593.]
4.6 Recombination
chiasmata (chapter 3)
physical manifestation of crossing-over
4.6 Recombination
chiasmata (chapter 3)
physical manifestationof crossing-over
help homologous chromosomes align at equator
4.6 Recombination
chiasmata (chapter 3)
are preceeded by DSB’s(double-strand breaks)
occur at “hot spots”certain positions where breaks are more like to occur
© 2006 Jones and Bartlett Publishers
Fig. 4.30. Molecular mechanism of recombination. [After D. K. Bishop and D. Zickler. 2004. Cell 117: 9.]
repair ?
4.6 Recombination
3’ broken end invades intact chromosomebase-pairs with complimentary strandother strand forms “D-loop”
3’ end is elongatedeventually ejected from template (?)
base pair with other end of break
fill in missing nucleotides
non crossing-over
4.6 Recombination
3’ broken end invades intact chromosomebase-pairs with complimentary strandother strand forms “D-loop”
D-loop expandsacts as template for other broken strand
base pair with other end of break
fill in missing nucleotides
crossing-over
© 2006 Jones and Bartlett Publishers
Fig. 4.31. Two Holliday junctions in a pair of DNA molecules undergoing recombination [EM, © 1997 from Essential Cell Biology, 1st Edition by Dr. Bruce Alberts. Reproduced by permission of Garland Science/Taylor & Francis Books, Inc.]
Holliday junction-resolving enzyme
=
12.3 Transposable elements
Discovered in corn by Babara McClintock(noble prize, 1983)
Pieces of DNA that could move around
Many encode their own transposase
12.3 Transposable elements
Different classes of transposons
DNA transposonsLTR retrotransposonsnon LTR retrotransposons
LINE longSINE short
interspersed elements
12.3 Transposable elements
DNA transposons(cut and paste transposition)
have terminal inverted repeatsbinding sites for transposase
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Fig. 12.8. Sequence arrangement of a cut-and-paste transposable element and the changes that take place when it inserts into the genome
LTR retrotransposons
long terminal repeats
direct repeats
same orientation
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Fig. 12.9. Sequence in direct and inverted repeats
LTR retrotransposons
long terminal repeats
direct repeats
inverted repeats
same orientation
inverse orientation
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Fig. 12.9. Sequence in direct and inverted repeats
LTR retrotransposons
long terminal repeats
direct repeatsinverted repeats
both use RNA intermediate(transcription, then RT)
© 2006 Jones and Bartlett Publishers
Fig. 12.10. Sequence organization of a copia retrotransposable element of Drosophila melanogaster
non-LTR retrotransposons
no long terminal repeats
long interspersed elementsshort interspersed elements
Alu I family300 bp106 copies11% Human DNA
Transposable elements
can cause mutations
insertion into a geneloss of function (knockout)
recombination between trans. elementsdeletions, inversions or duplications
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Fig. 12.11. Recombination between transposable elements in the same chromosome
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Fig. 12.12. Unequal crossing-over between homologous transposable elements present in the same orientation in different chromatids
Transposable elements
make up much of the human genome (45%)
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Table 12.2. Transposable elements in the human genome
Transposable elements
make up much of the human genome (45%)
function ?
SINEs transcribed under stressmariner horizontal transmission
(species to species)