Download • Will a base pair substitution, addition or deletion cause a

2/19/10 MUTATION JARGON
• Will a base pair
substitution, addition
or deletion cause a
phenotype?
• Will a larger scale
change such as a
gene duplication
cause a phenotype?
• What information do
you need to address
this question?
Science Dec. 2007
http://www.sciencemag.org/cgi/content/full/318/5858/1842
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Woe to that child which when kissed on the
forehead tastes salty. He is bewitched and soon
must die. This adage, from northern European
folklore, is an early reference to the common genetic
disease recognized today as cystic fibrosis. As the
saying implies, the disorder once routinely killed
children in infancy and is often identifiable by
excessive salt in sweat..
(Scientific American Dec. 1995)
cystic fibrosis: most common severe recessive
monogenic disorder affecting people of European
descent
Info about cystic fibrosis
http://www.nlm.nih.gov/medlineplus/cysticfibrosis.html
http://ghr.nlm.nih.gov/condition=cysticfibrosis
http://www.ygyh.org/
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the “cystic fibrosis” gene codes for a transmembrane protein
involved in chloride transport
(note gene is named for its mutant phenotype and not for the
protein that it specifies)
CFTR= cystic fibrosis transmembrane conductance regulator
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• All individuals with sickle cell anemia have the same
missense mutation in the B globin gene.
• In contrast, over 1600 different mutant alleles of the CF gene have
been discovered world-wide
Cystic Fibrosis Gene: CFTR
http://www.genet.sickkids.on.ca/cftr/StatisticsPage.html
see statistics, gene, consortium data
search database: pull up gene map
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CF mutations are distributed throughout the gene
http://www.genet.sickkids.on.ca/cftr/PicturePage.html
Sequence variation = non-disease-causing. It is sometimes designated as
“polymorphism”, According to the definition on this web site, a “sequence
variation” has to reach an allele frequency of 1% to be a polymorphism. When a
sequence variation is found in only one single individual, it is not possible to
determine if it is a “non-disease-causing” variation.
Retrieval of Genetic Information: Central to any information storage system is the ability to
access and retrieve the information and to convert it to a usable form. In addition to the
sequence information that will be translated into protein via the triplet code, a gene also contains
sequence information that specifies
1. where transcription starts and stops on a given stretch of DNA and which strand of DNA
is transcribed
2. where splicing occurs (exon/intron boundaries)
3. where, when and at what level the transcript will be produced
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find most common mutation:
http://www.genet.sickkids.on.ca/cftr/ConsortiumDataPage1.html
http://www.genet.sickkids.on.ca/cftr/resource/Table1.html
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M UTATION J ARGON
POINT MUTATION or GENE MUTATION
Scale of mutation is small
Alteration of a single base pair or a small number of adjacent base pairs
↓
AT THE DNA LEVEL
 single base pair substitutions: transitions & transversions
 indels: insertion or deletion of a few base pairs
 other: transposon insertion
AT THE LEVEL OF
GENE EXPRESSION
AT THE PROTEIN
LEVEL
promoter mutations
splicing mutations
regulatory mutations
nonsense
missense
[neutral]
silent
frameshift
AT THE LEVEL OF GENE FUNCTION
loss-of-function
gain-of-function
[neutral]
↑
CHROMOSOME MUTATION
involves segments of chromosomes or whole chromosomes
CNVs = copy number variations
alterations in chromosome structure and number
(deletions, duplications, translocations and inversions)
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DNA
TCA
5'
3'
AGT
transcription
TCA
5'
3'
5'
3'
3'
UCA
5'
AGT
splicing and processing
in eukaryotes
serine codon
on mRNA
mRNA
serine anticodon
on tRNA
UCA
AGU
3'
5'
5'
serine attached to
tRNA ser at 3' end
serine
Chemical conversion of TCA into serine.
Accuracy of translation depends on precise matching:
(1) of an amino acid with its cognate tRNA
(2) of the anitcodon of a charged tRNA with its corresponding
codon on the mRNA
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NOTE: code is always in RNAspeak
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What is a missense mutation?
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Missense mutation:
a mutation that alters a codon so
that a different amino acid is
specified
How will any given missense mutation
affect the functioning of a protein?
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Hard to say a priori without additional
information on:
•
•
the nature of the amino acid substitution
the site of the mutation in the protein
A missense mutation may
1. have virtually no affect on protein function –
especially if a chemically similar amino acid is
substituted
2. partially or completely inactivate the protein
• if the amino acid substitution is in the active site or another
site critical for function
• if the mutation affects the folding or stability of the protein
• if the mutation affects the processing of the protein or
interferes with its transit to the appropriate cellular
compartment. See interesting example: In Sex Reversal,
Protein Deterred by Nuclear Barrier
http://fire.biol.wwu.edu/trent/trent/sexreversal.pdf
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Recent study on a protein called human factor VIII
which has a critical role in blood clotting
(Nature November 25, 1999)
• Factor VIII is a glycoprotein that has a critical role in blood
coagulation
• This protein circulates as a complex with other proteins
• Gene coding for clotting factor VIII is mutated in the Xlinked disease state hemophila A
21 different amino acid residues in factor VIII are
known to be sites of deleterious mutations in patients
with hemophila
• A number of these are in the hydrophobic protein core
• other mutated amino acids are involved in hydrogen bonding
networks that clearly stabilize protein folding
• still others are on the exposed surface of the protein and
presumably are important for the interaction of factor VIII
with other proteins
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The enzyme lactate dehydrogenase catalyses the
following reaction:
pyruvate + NADH  lactate the NAD+
What would the effect be of substituting a different amino acid for
arginine?
14
Neutral Mutation:
• a mutation that has no effect on the Darwinian
fitness of its carrier: an allele that has a
negligible effect on the ability of the organism to
survive and reproduce
Neutral Missense Mutation:
• a subset of missense mutations in which the
effect of the amino acid change on protein
function is negligible or is not deleterious to the
organism
for example:
AGA arg  AAA lys
both are basic amino acids: substitution of arg for lys
may not affect protein function
arg = arginine lys = lysine
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BUT: don’t assume that a chemically equivalent
substitution will always be neutral
Example:
Protein: Triose-P-isomerase
Glu  Asp change in active site decreases catalytic
activity 1000X
glu= glutamic acid
asp = aspartic acid
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Silent (same sense) Mutation:
a mutation that doesn’t change the meaning of
the codon (the same amino acid is specified)
CUU (leu)  CUC (leu)
Degenerate code:
many amino acids are specified by more
than one codon
17
Nonsense mutation: ?
18
Nonsense mutation: a mutation that
generates an abnormal stop codon
It results in premature termination of
translation and a truncated polypeptide
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Reading Frame: the codon sequence that
is determined by reading nucleotides in
groups of 3 from some specific start
codon (AUG)
How does the translation machinery know where
to start?
How does a eukaryotic ribosome identify
the correct reading frame?
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Frameshift mutatio ?
21
Frameshift mutation:
addition or deletion of one or a few base pairs
causing a change in the translational reading
frame
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BRCA1 database
BRCA1 gene plays a role in the repair of double-stranded
breaks in DNA
About 5% of breast cancer cases are caused by an inherited susceptibility allele. A
mutation in a gene called BRCA1 is thought to account for approximately 80% of
families with a high incidence of both early-onset breast and ovarian cancer. Table
1 summarizes some of the data that has been collected on BCRA1 mutations in
such families. Table 2 shows neutral polymorphisms found in control families (not
showning an increased frequency of breast and ovarian cancer).
Table 1. Predisposing mutations in BRCA1. Science 266: 66 1994
NA indicates not applicable. ND = not determined
1901
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Mutation
Nucleotide
change
-11 bp
2082
1910
2099
2035
1313
1756
1775
NA
Extra C
T-->G
ND
Kindred
Codon #
Coding
effect
frameshift
Gln --> Stop
frameshift
Met --> Arg
Loss of
transcript
Table 2.
Frequency
in control
chromosomes
0/180
0/170
0/162
0/120
ND
Neutral polymorphisms in BRCA1. For the frequency in control chromosomes, the
number of chromosomes with a particular base at the indicated polymorphic site is shown
(A,C,G, or T).
Frequency in control
chromosomes
Codon
Base
Name
location in codon*
A
C
G
T
PM1
317
2
152
0
10
0
PM6
878
2
0
55
0
100
PM7
1190
2
109
0
53
0
PM2
1443
3
0
115
0
58
PM3
1619
1
116
0
52
0
*That is, position 1,2 or 3 of the codon.
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How do point mutations affect the functioning of a gene?
DNA
RNA
PROTEIN
Information Contained
in the Sequence of a Gene
Proper functioning
of a gene requires:
1. Coding Region
1. An intact gene product
(protein or RNA)
specifies RNA & amino acid sequence
2. Other Sequence Information
2. Proper expression of the
gene:
(signals for generating RNA)
a. promoter (RNA polymerase
binding site)
transcription termination site
a. transcript generated from
the correct stretch of DNA
b. regulatory elements
(operators in prok's;
enhancers in euk's)
b. transcript generated
in the appropriate amount
at the appropriate time
in the appropriate cells
c. transcript spliced correctly
c. splice site signals
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