© 2006 Jones and Bartlett Publishers Chapter 12 Opening photo. Peacock [© Photos.com]

© 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

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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

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Fig. 8.23. Reading bases in an RNA molecule

codons are linear and non-overlapping

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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

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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

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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

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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

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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)

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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

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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

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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

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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

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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

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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

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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)

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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)