Transcript Sect7Mutation

This presentation was originally prepared by
C. William Birky, Jr.
Department of Ecology and Evolutionary Biology
The University of Arizona
It may be used with or without modification for
educational purposes but not commercially or for profit.
The author does not guarantee accuracy and will not
update the lectures, which were written when the course
was given during the Spring 2007 semester.
Introduction
• How do we detect genes?
1. Detecting phenotypic changes in the organism due to changes in the gene.
2. Screening DNA sequences for ORFs with gene-like features or similarities
to genes already known.
• A mutation is a change in base sequence of a gene, or in the arrangement of
genes on a chromosome (chromosome mutation, discussed more later).
• A mutation produces a new allele of the gene.
• The most common allele in a laboratory stock or wild population of an
organism is called the wild type allele; then a mutation produces a new
mutant allele.
• A mutant is an individual carrying a mutant allele.
Wild type Drosophila on the left; each of the three mutants is in a different gene.
Some Kinds of Mutations
wild type
ATGACCATGA
base pair substitution
AGGACCATGA
AAGACCATGA
ACGACCATGA
insertion
ATGGACCATGA
ATGTTACCATGA
ATGTTCACCATGA
Deletion
dT
AGACCATGA
dTG
AACCATGA
dTGA
ACCATGA
• Changes of 1 bp (or a few contiguous bp) are called point mutations.
• Also possible but much less frequent are substitutions of ≥ 2 bp at one
time.
• Transposable elements cause insertions of 102 - 104 bp.
Mechanisms of Point Mutation (partial list)
1. Most are due to errors in DNA replication:
replication
repair
GATC --> GATC --> GATC
CTAG
CCAG
CTAG
or GGTC
CCAG
Repair occurs during proofreading or later.
Slip-strand mispairing causes short repeats: NNNNNNAGCAGCAGC … NNN
e.g. Huntington’s disease, (AGC)n, an in-frame repeat encoding poly(Glu). The
resulting polypeptide causes cell death in parts of the brain and dominant
neurological problems. (N stands for nucleotide, i.e. any base.)
2. Some are due to spontaneous changes in bases, e.g. C --> deamination --> U
Mechanisms of Point Mutation (continued)
H. J. Muller
3. Some mutations are caused by mutagens
Chemical mutagens modify bases so they cause mispairing when replicated or
are repaired incorrectly.
UV radiation links adjacent pyrimidines to form dimers, which may be repaired
incorrectly.
Ionizing radiation induces single- or double-stranded breaks, chemically
modifies bases, or cross-links bases; may be repaired incorrectly.
When and Where Mutations Happen
Mutations are stochastic events (unpredictable, random): we can never predict exactly
when a mutation will occur or what kind of mutation will occur, but we can assign a
probability (frequency or mutation rate) to it.
Most mutations happen during cell division, so we usually measure the rate in mutations
per cell division.
Measured mutation rate would be 7 mutations/31 cell divisions or 0.226 mutations/cell division
(unrealistically high example!).
When and Where Mutations Happen
In eukaryotes, mutations that occur in the somatic cells (somatic mutations) are not
inherited; mutations that occur any time in the germ line are inherited. We usually measure
the rate in mutations per gamete
somatic mutation
Soma
Germ line
germ line mutation
Mutation Rates
Mutations are stochastic events (unpredictable, random): we can never predict exactly when
a mutation will occur or what kind of mutation will occur. We can measure the rate at which
a given kind of mutation will occur. The rate is the probability that it will occur in a unit of
time. Orders of magnitude:
animals and plants
10-6 - 10-4 muts per gene per gamete
10-10 - 10-8 muts per bp per year (from evolution rates)
10-9 muts/bp  year  3  10 9 bp > 1 new mutation in each gamete (we are all mutants!)
Drosophila: u = 8.4  10-9 muts/bp  year U = 1.2 detrimental mutations/2N genome
bacteria
10-10 - 10-6 muts per gene per cell division
How to write rates so they can be used in dimensional analysis:
10-9 muts per bp per year
10-9 muts/bp  year
NOT 10-9 muts/bp/yearbecause that is 10-9 muts  year /bp
Mutagenesis increases the rate (probability) but the mutations are still stochastic.
Mutation Rates
A given mutagen increases the rate of some kinds of mutations but not others. In other
words, it biases the probabilities, like weighting a coin.
e.g.
• Ionizing radiation induces (increases the probability of) chromosome breaks and hence of
large-scale “chromosome mutations” or rearrangements such as deletions or
translocations.
• Acridines induce deletion or insertion frameshifts.
• Ethyl methan sulfonate (EMS) induces chemical changes in bases, mainly changing
guanine to O6-ethylguanine which mispairs, resulting in changing GC pairs to AT.
• 5-bromouracil (Bu) is an analogue of thymine and is most often incorporated into DNA in
place of thymine. It then pairs with G at the next replication, so that an AT pair is
replaced by a GC pair. Less commonly it causes a GC pair to be replaced by AT.
Modern genetic engineering can reduce or eliminate the stochasticity of mutation, allowing
us to change a specific base in a specific gene in vitro, then put it back in the organism.
Knockout mutations can be made in specific genes in vivo.
Phenotypic Effects of Mutations in Exons
Review code properties:
• Degenerate
• Triplet
• Commaless
• Start codon
• Stop codon(s)
Phenotypic Effects of Mutations
Useful terms for phenotypes of mutations:
Amorph = nullimorph = null = knockout mutations: mutant allele is
completely inactive (not transcribed, or translated, or encodes inactive
protein or RNA).
e.g. white eye mutant in Drosophila melanogaster
Hypomorph: mutant allele has reduce activity (reduced rate of
transcription or translation, or encodes protein or RNA with reduced
activity).
e.g. apricot eye allele of white gene in D. melanogaster
Neomorph = gain-of-function mutation: mutant allele has new activity
(e.g. encodes protein or RNA with new enzymatic activity or turns one
gene off and another one on or make a gene active in wrong tissue).
e.g. Antennapedia in Drosophila
The “morph” terminology was devised by H. J. Muller, who used it
mainly to refer to mutation effects inferred from phenotypic effects.
Qui ckTime™ and a
TIFF (LZW) decompr essor
are needed to see this pictur e.
Consequences of Code Properties
Degenerate
Consequences of Code Properties
• Degenerate, therefore in an
exon of a protein coding
gene there are two kinds of
point mutations:
Synonymous mutations
change a codon to a
synonymous codon and do
not change an amino acid
Nonsynonymous =
missense mutations
change an amino acid
Consequences of Amino Acid Changes
Synonymous mutations do not change an amino acid.
Therefore they usually have no effect on phenotype.
Nonsynonymous = missense mutations change an
amino acid. The phenotypic effect depends on the
nature of the change and the location in the protein:
Some changes have no effect on protein function,
therefore no effect on phenotype.
Changes that are more likely to have an effect include:
•Changing to amino acid with very different side chain
(charge, hydrophobicity, etc.).
•Change in active site or ligand binding site or site
required for folding or joining with other polypeptides.
•Changes on outside of molecule that affect
hydrophobicity or charge of protein.
Hemoglobin. Heme in red, two
a and two b chains in different
colors.
Phenotypic Effects of Mutations
• Triplet
• Commaless
Phenotypic Effects
of Mutations
• Triplet
• Commaless
Deletions of 1 or 2 bp are frameshift mutations.
Consequences:
Met
Val
His
Leu
Thr
Pro
Glu
His
Term
CACCATGGTGCACCTGACTCCTGAG…CACUAAGCU
Met
Val
Thr
Term
CACCATGGTGACCTGACTCCTGAG…CACUAAGCU
Met
Val
-C
Pro
Pro
Asp
Ser
Leu
Thr
Arg
Term
CACCATGGTGCCACCTGACTCCTGAG…CACUAAGCU
+C
Met
Val
His
?
Leu
Ser
CACCATGGTGCACCTGACTCGAG…CACUAAGCU
- CT
Phenotypic Effects
of Mutations
Deletions of 1 or 2 bp in exons are frameshift
mutations.
Consequences:
• Change of one or more amino acids; phenotype
depends on the amino acids and their location.
• Premature termination.
• Late termination.
• Usually nullimorphs or hypomorphs.
Met
Val
His
Leu
Thr
Pro
Glu
His
Term
CACCATGGTGCACCTGACTCCTGAG…CACUAAGCU
Met
Val
Thr
Term (termination, or stop)
CACCATGGTGACCTGACTCCTGAG…CACUAAGCU
Met
Val
-C
Pro
Pro
Asp
Ser
Term
CACCATGGTGCCACCTGACTCCTGAG…CACUAAGCU
+C
Met
Val
His
Leu
Thr
Arg
?
Leu?
Ser?
CACCATGGTGCACCTGACTCGAG…CACUAAGCU
- CT
Phenotypic Effects of
Mutations in Exons
• Start codon
• Stop codon
Copyrighted figure removed.
Phenotypic Effects of
Mutations in Exons
Any change in start codon
• Late start, first part of polypeptide missing.
• Out-of-frame start.
Likely result is polypeptide with no function
(nullimorph) or abnormal function (hypomorph).
Copyrighted figure removed.
Change of stop codon to a sense codon
• Polypeptide has extra segment at the end.
Possible result is polypeptide with no function, reduced
function, or abnormal function (amorph, hypomorph, or
neomorph).
Nonsense mutations change a sense codon for an amino
acid to a nonsense = stop codon.
• Shortened polypeptide which usually, but not always, has
reduced or no function.
Mutations Due to Transposable Elements
Transposable elements (TEs) are segments of DNA that can move from one location
to another in the genome, or have a copy made and moved to a new location.
Insertion of a TE in a gene can cause frameshifts or cause additional amino acids to
be added to a protein product. This usually results in a null mutation.
Insertion of TEs in controlling elements or between a gene and its controlling
elements can cause major changes in transcription (no transcription or transcription
at inappropriate times and/or places.
Transposable elements are more common in some organisms (e.g. Drosophila
melanogaster) than in others and can cause a major proportion of all visible
mutations.
Genes Encoding Enzymes and Auxotrophic Mutants
Some genes code for enzymes.
Many enzymes catalyze steps in biosynthetic pathways.
Steps are sequential, enzymes act sequentially.
Usually, 1 enzyme catalyzes 1 reaction or step in a pathway.
Genes
G1
G2
G3
Enzymes E1
E2
E3
A
B
C
D
Chorismic acid
e.g. tryptophan biosynthesis in E. coli:
Text says 5 steps but don’t show the pathway.
Notice that names of genes are italicized
(or underlined).
Anthranilic acid
ASase
trpE
PRTase
trpD
InGPSase
trpC
TSaseB
trpB
TSaseA
trpA
PRA
CDRP
InGP
Indole
L-Tryptophan
Genes Encoding Enzymes and Auxotrophic Mutants
Special classes of mutants especially useful with microorganisms:
Antibiotic-resistant
Auxotrophic: can’t make essential nutrient, must be supplied in culture
medium
•Test for growth on minimal medium (MM) which has things wild type
needs (energy, C, N sources; salts; etc.); only wild type (prototroph)
grows, because it can make everything it needs from these simple
ingredients.
•Control is complete medium (CM) which has yeast extract, proteose
peptone, etc. that supply everything; all genotypes grow.
One Gene - One Enzyme Hypothesis: not quite right, but focused on
idea that genes have their phenotypic effects by encoding proteins.
Tryptophan Auxotrophs
Chorismic acid
Anthranilic acid
ASase
trpE
PRTase
trpD
InGPSase
trpC
TSaseB
trpB
TSaseA
trpA
PRA
CDRP
InGP
Indole
L-Tryptophan
+
trp
trpA
trpB
trpC
trpD
trpE
Grows
On
CM MM MM+Trp MM+indole MM+InGP MM+CDRP
+ +
+
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+
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Visible Phenotypes of Pathway Mutants
cinnabar
Drosophila melanogaster wild type has red eyes, which require a number
of pigments.
Makes eye pigment xanthommatin from tryptophan.
cinnabar (cn+) gene encodes kynurenine-3-hydroxylase.
cinnabar (cn) mutant has cinnabar-colored eyes.
Phenotypic Effects of Mutations in Introns
Usually no effect unless mutation in splicing signal sequence --> failure to remove intron
or splicing at incorrect site.
ATG GTG CAC … GCAGgttggtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcatgtggag
AcagagaagactcttgggtttctgataggcactgactctctctgcctattggtctattttcccacccttagGCTGCTG
GTGATGGTGCAC … GCAGGCTGCTGG
DG
No splicing, wrong amino acids up to a stop codon
ATG GTG CAC … GCA Ggt ggt atc aag gtt aca aga cag gtt taa gga gac
caa tag aaa ctg ggc atg tggagacagagaagactcttggg ttt ctg
ata ggc act gac tct ctc tgc cta ttg gtc tat ttt ccc acc ctt agG
CTG CTG GTG
Phenotypic Effects of Mutations in Genes
Encoding Functional RNAs
rRNA genes
Complicated because rRNA genes present in hundreds or thousands of copies (tandem
repeats); mutation affects only one, in which case has little or no effect, but can spread
to all copies after many generations. The mechanism of spreading is unequal crossingover and gene conversion, which we will discuss later in the course.
If it spreads, can have no effect, nonfunctional or subfunctional RNAs and ribosomes,
no or reduced synthesis of all proteins encoded same genome. Often lethal or very
detrimental.
Phenotypic Effects of Mutations in Genes
Encoding Functional RNAs
tRNA genes
Mutations in the anticodon, or in the region that is recognized by the aminoacyl tRNA
synthetase, can result in wrong amino acids being inserted at many sites in many
proteins.
Mutations and Gene Interactions
mRNA
Gene A
protein
Gene B
Control of gene expression involves interaction with the products
of other genes. (More detail in next section of lectures.)
• Sometimes, whether a gene is transcribed or not depends on whether
or not a protein produce of another gene binds to an upstream
controlling region.
• Mutations in controlling gene can modify presence/absence or
abundance of product of controlled gene.
• There are many other ways in which products of different genes
interact.
• Consequence: change in expression of a gene may be due to
mutation in gene itself, or in its control sequences, or in other genes
that interact with it.
• What appears to be a mutation in gene B could be a mutation in gene
A if A encodes a protein that is required for transcription of B.
Dominance
An important aspect of genetics is being able to relate the genotype
with respect to a particular gene to the phenotype.
Dominance: allele A1 is dominant to allele A2 if the heterozygote
A1/A2 has the same phenotype as the homozygote A1/A1.
Dominance of a mutant allele depends on the effects of the
mutation at the molecular level.
e.g. a null alllele will be recessive to the wild type in a gene that
codes for a product that is detected fairly directly.
You are invited to try to predict the phenotypes of heterozygotes
for various kinds of mutations.
Mutation Summary
Mutations can result from:
• errors in DNA replication or repair
• damage by chemical or radiation mutagens
• movement of transposable elements
The effects of mutations depend on:
• the kind of gene in which the occur
• the function (or lack thereof) of the site within the gene
• the nature of the mutation
• interactions of the mutant gene or sequence with other genes