Download GENE MUTATION = POINT MUTATION at the DNA level: at the level

EFFECT OF MUTATIONS ON PROTEIN FUNCTION
GENE MUTATION = POINT MUTATION
(scales of mutation is small and is localized to a specific region,
a single nucleotide or a few adjacent base pairs)
↓
at the DNA level:
 single base pair substitutions: transitions & transversions
 single (or a few) base pair addition or deletion: indels
 gene mutation by transposon insertion
at the level of
gene expression:
at the protein
level:
promoter mutations
splicing mutations
regulatory mutations
nonsense
missense
[neutral]
silent*
frameshift
*aka synonymous
at the level of gene function:
loss-of-function
gain-of-function
[neutral]
1
CHROMOSOME MUTATION
• involves segments of chromosomes or
whole chromosomes or whole genomes
• alterations in chromosome structure
and number
• deletion, duplications, translocations
and inversions
• CNVs: copy number variations
NOTE: you are responsible for frameshift,
nonsense and synonymous(silent)
mutations even though we will not cover
these terms in class.
2
Finding your way around a eukaryotic gene
 upstream = 5’ of….
downstream = 3’ of… 
3
Conventions for displaying gene sequences:
• Only the mRNA-like strand is
displayed (complementary strand not
shown)
• Sequence reads 5’ to 3’
• A cDNA* sequence will reflect the
sequence of the spliced mRNA and
will therefore not include intron
sequence
• A genomic sequence will include
introns and exons and adjacent regulatory regions – sometimes the
introns will be indicated in lower case and the EXONS in
uppercase (see pg 8 of this lecture)
* see figure 10-4 in text: Double-stranded DNA is synthesized from mRNA
4
Genomic DNA sequence display
LOCUS
NG_011751
7897 bp
DNA
linear
PRI 05-FEB-2012
DEFINITION Homo sapiens sex determining region Y (SRY),
RefSeqGene on chromosome Y.
TGACCTTCATTTTATGGAGAGAAACAAGCTATAACATGTAGTATCTAAGCTGATTAGAAGAACTAAAAAG
AGAAGCTCATACTTGTGCATCAGAAGGTAAATGAAAGAGTGAAGTTACCTCTTTGTTTTAAGGAAGAAAG
GAAAATTGTGGATGTCATCTGTTTTCTGTTTACATATTTCAGGCATGGATAGCCACAATGTGATTTTAAG
ACGGTTAGTTACAACTGATTTGAAAAAAAAAAAAAATGCTTCACTCTATGAGAAATTTCTTCCCAAGTAT
GAAACCTTGTTTTTACAGGCAATTTCCTATACTTTGAAAAAATCAAAATAATAAAGTAAAAGAAAAATAA
TTCAGGTGAAGTTAGAGAAAAAAACAGGCAGCATTATTTTAAAGTTGTAAACTATTTTGTTTACTTATAG
TTTAATTTACATGTAGTAGATATGCATTTGTAAGGTTCTTCGGCTCAGGTAGGAGATCATTCTATTTCCC
ACTGCACCCTACTTCATCCTCCCACTGGCAAATAATTAGATTATCCCTGGGAAAAAAAGATGCCAGTAAA
ATTGATCATGTTTAAATGCATCAGTTGCTAGGTGATTTATCTGATTAAGTCTTGAAACAGTAGAACCTAG
CAATTAAAGTGAGCATTAACTTCTACCTACCAAATCAGAAGACTATTCTAACTTTTTGAGAATTAGATGT
TGAAAATATGGCCCATGAATTTAGCATGGTTAAAATAAAAAACATGCAAACAAAACAAACCCAACATCTT
GAAAGGACATTTGACTCTAAAGTCCCAAAAATAATCACAAGTCTAAAAATCCTAAGTTTAGTGTTACTCT
ATTACACCTTTTTATTTGTAAGTGTCCTTTCACAAAAGTTTTAAATTTTGCTCTTGTGCATTTTATTTAC
CTTTTCTTTTGTTGTTTGTGTCTTTGGTGACCTGCCAACCATTAGACTTCAAAAAACAGCCTATAGCCAA
GCTGCAGGATAAATGAACACATAAGTTGACTTAGAATAGTCAACTCTGTCTAGTATACAATTTATGGGGG
ATGGTTTATGACCACATATATTTCTACTTTGATGGGAATATCTTGAGATAAAATTAGAGAGAATGAGTGG
AGTAATATTCACAACATTTTTGCTGCATTCATCCCTGAATTTGAAGAAATACCAAAGTACATCTTGTGAG
GAGAAAAAATAAATAAATTCATATAAAATGTTGTGGGTTTTATTCTTTATGCAGTGGTAAACTGTGTTTG
CATACACCATAGCAATTAAATTAGGGCTACAAAGGGTATTTAACTAATGAGCATAAAATACCTTAATGTA
CCTCAAATGCAATTAATTGCATTGGACCAATCTAAGTTACTATTCTTCAGTTTTCATTTTTATTTCATTA
TTCATTTCATTTTTATTCTGATATAAAAATGAACCAGGATCTGTGTGAAATTATTTGAATCTAATGTCTT
TGAACATTTTTCTTACCATACCTTAAGATTAAAAAAACAAAAAAAAATCCCTTAGTTTGGCAACTTTTGC
TGTTGGTTAAGCCCGTTTGGATTTAACATTGACAGGACCAGCTAACTTCCTACCAGTTAACATTGCTTGT
…………… etc
5
cDNA/mRNA sequence display
LOCUS
NM_003140
897 bp
mRNA
linear
PRI 17-DEC-2011
>gi|4507224|ref| Homo sapiens sex determining region Y (SRY), mRNA
GTTGAGGGGGTGTTGAGGGCGGAGAAATGCAAGTTTCATTACAAAAGTTAACGTAACAAAGAATCTGGTA
GAAGTGAGTTTTGGATAGTAAAATAAGTTTCGAACTCTGGCACCTTTCAATTTTGTCGCACTCTCCTTGT
TTTTGACAATGCAATCATATGCTTCTGCTATGTTAAGCGTATTCAACAGCGATGATTACAGTCCAGCTGT
GCAAGAGAATATTCCCGCTCTCCGGAGAAGCTCTTCCTTCCTTTGCACTGAAAGCTGTAACTCTAAGTAT
CAGTGTGAAACGGGAGAAAACAGTAAAGGCAACGTCCAGGATAGAGTGAAGCGACCCATGAACGCATTCA
TCGTGTGGTCTCGCGATCAGAGGCGCAAGATGGCTCTAGAGAATCCCAGAATGCGAAACTCAGAGATCAG
CAAGCAGCTGGGATACCAGTGGAAAATGCTTACTGAAGCCGAAAAATGGCCATTCTTCCAGGAGGCACAG
AAATTACAGGCCATGCACAGAGAGAAATACCCGAATTATAAGTATCGACCTCGTCGGAAGGCGAAGATGC
TGCCGAAGAATTGCAGTTTGCTTCCCGCAGATCCCGCTTCGGTACTCTGCAGCGAAGTGCAACTGGACAA
CAGGTTGTACAGGGATGACTGTACGAAAGCCACACACTCAAGAATGGAGCACCAGCTAGGCCACTTACCG
CCCATCAACGCAGCCAGCTCACCGCAGCAACGGGACCGCTACAGCCACTGGACAAAGCTGTAGGACAATC
GGGTAACATTGGCTACAAAGACCTACCTAGATGCTCCTTTTTACGATAACTTACAGCCCTCACTTTCTTA
TGTTTAGTTTCAATATTGTTTTCTTTTCTCTGGCTAATAAAGGCCTTATTCATTTCA
A sequence logo showing the most conserved bases around the initiation codon from all human mRNAs. The
larger the LETTER at a given location, the greater the importance of a the specific base
6
LOCUS
NP_003131
204 aa
linear
PRI 17-DEC-2011
>gi|4507225|ref| sex-determining region Y protein [Homo sapiens]
MQSYASAMLSVFNSDDYSPAVQENIPALRRSSSFLCTESCNSKYQCETGENSKGNVQDRVKRPMNAFIVW
SRDQRRKMALENPRMRNSEISKQLGYQWKMLTEAEKWPFFQEAQKLQAMHREKYPNYKYRPRRKAKMLPK
NCSLLPADPASVLCSEVQLDNRLYRDDCTKATHSRMEHQLGHLPPINAASSPQQRDRYSHWTKL
Amino acid sequence reads from the N (amino) to the C (carboxyl) terminus
7
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/
8
the “cystic fibrosis”
gene codes for the
CFTR protein which is
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
9
http://www.genet.sickkids.on.ca/cftr/GenomicDnaSequencePage.html
10
http://www.genet.sickkids.on.ca/cftr/MRnaPolypeptideSequencePage.html
11
The first questions a researcher interested in exploring the molecular
genetics of a disease state addresses generally are
1. Does everyone affected with the disease have a mutation in the
same gene – in other words, is the disease genetically
heterogeneous?
2. For a given gene, what is the mutational spectrum for individuals
with this disease—does every affected person have the same
mutation or are there lots of different mutations?
3. How are the mutations distributed in the gene and how do they
affect gene function?
Cystic fibrosis is not genetically heterogeneous but it shows extensive
allelic heterogeneity
• Only mutations in the CF gene (see next page) cause CF, BUT over
1900 different mutant alleles of the CF gene have been discovered
world-wide
• In contrast All individuals with sickle cell anemia have the same
missense mutation in the B globin gene.
12
http://www.genet.sickkids.on.ca/cftr/StatisticsPage.html
13
CF mutations are distributed throughout the gene
http://www.genet.sickkids.on.ca/cftr/PicturePage.html
14
What does
G551D
mean?
G= glycine
D= Aspartic
acid
551= AA
position
New York Times 1/31/12
15
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
16
DNA
NOTE: code is always in
RNAspeak
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
serine attached to
tRNA ser at 3' end
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
http://en.wikipedia.org/wiki/Genetic_code
17
http://en.wikipedia.org/wiki/File:GeneticCode21-version-2.svg
18
What is a missense mutation?
19
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?
20
Hard to say a priori without additional information on:
•
•
•
the nature of the amino acid substitution
the site of the mutation in the protein
whether the change is in a highly conserved amino acid
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
• the mutation affects the folding or stability of the protein
• 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
3. result in a gain of gene function (see cancer genetics lecture)
21
A protein called human factor VIII 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 X-linked 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
22
A "ribbon diagram" of the
structure of the hemophilia
domain of human factor VIII
• In this figure, the positions in the
protein fold that are found to be
mutated in patients with hemophilia A
are shown by spheres.
• Dark spheres are sites that display
severe defects in clotting when
mutated
• Light spheres are sites that display
milder defects.
• The atoms at the bottom of the protein
are amino acids thought to embed
themselves into exposed membranes
at sites of blood-vessel damage.
http://depts.washington.edu/mednews/research/hemop
hilia.html
23
The enzyme lactate dehydrogenase catalyses the following reaction:
pyruvate + NADH  lactate the NAD+
Fig. 1. A cartoon of the active site of
lactate dehydrogenase showing the relative
arrangement of reacting groups (not to
scale). The substrate pyruvate is shown;
the ‑CH3 group is replaced by ‑NH2 to form
oxamate. The hydride transfer is indicated
by the bold arrow, hydrogen transfer by
light arrow.
http://www.bioc.aecom.yu.edu/labs/calllab/highlights/LDH.htm
24
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:
codon AGA specifies arg  codon AAA specifies lys
arg = arginine lys = lysine
both are basic amino acids: substitution of arg for lys may not affect
protein function
25
Don’t assume that a chemically equivalent substitution will be neutral
Protein: Triose-P-isomerase
Glu  Asp change in active site decreases catalytic activity 1000X
glu= glutamic acid
asp = aspartic acid
26
Missense mutations in conserved amino acids
Nature Genetics VOLUME 43 | NUMBER 6 | JUNE 2011
Mutations in DNMT1 cause hereditary sensory neuropathy
with dementia and hearing loss
Neurodegenerative diseases are hallmarked by an increased rate of neuronal
axonal loss. Neural development, neural survival and connectivity have been
linked to DNA methylation and chromatin stability. Here, we studied one form
of neurodegeneration with both peripheral and central involvement: hereditary
sensory and autonomic neuropathy type 1 (HSAN1) with dementia and hearing
loss.
DNA methyltransferase 1 (DNMT1) is crucial for maintenance of
methylation, gene regulation and chromatin stability1–3. DNA mismatch repair,
cell cycle regulation in post-mitotic neuron and neurogenesis are influenced by
DNA methylation. Here we show that mutations in DNMT1 cause both central
and peripheral neurodegeneration in one form of hereditary sensory and
autonomic neuropathy with dementia and hearing loss.
27
Figure 2 DNMT1 mutations in studied HSAN1 kindreds with dementia and hearing loss. (a–d)
Pedigrees of four kindreds are shown. We identified a heterozygous mutation, c.1484A>G, resulting
in p.Tyr495Cys, in exon 20 of DNMT1 in the HSAN kindred 1 (a), 3 (c) and 4 (d). We identified
three consecutive heterozygous mutations, c.1470–1472TCC>ATA, resulting in the p.Asp490Glu–
Pro491Tyr substitutions, also in exon 20 of DNMT1, in the HSAN kindred 2 (b). We sequenced all
available samples (asterisk) from these four kindreds to confirm that the mutation segregated with
disease.
28
29
Figure 2 DNMT1 mutations in
studied HSAN1 kindreds with
dementia and hearing loss. (a–d)
Pedigrees of four kindreds are
shown. We identified a
heterozygous mutation, c.1484A>G,
resulting in p.Tyr495Cys, in exon
20 of DNMT1 in the HSAN kindred
1 (a), 3 (c) and 4 (d). We identified
three consecutive heterozygous
mutations, c.1470–1472TCC>ATA,
resulting in the p.Asp490Glu–
Pro491Tyr substitutions, also in
exon 20 of DNMT1, in the HSAN
kindred 2 (b). We sequenced all
available samples (asterisk) from
these four kindreds to confirm that
the mutation segregated with
disease. (e) Schematic overview of
DNMT1 and its multiple domains in
the N-terminal region. PBD, PCNAbinding domain; TS, targeting
sequence; ZnF, zinc finger; BAH1
& 2, bromo adjacent homology domains 1 and 2. Below is the ClustalW alignment of the part of the targeting sequence domain where
mutations occur from multiple DNMT1 homologs. Comparison of human DNMT1 (P26358) and its orthologs in mouse
(P13864), rat (Q9Z330), cow (Q24K09), sheep (Q865V5), zebrafish (Q8QGB8), frog (Q6GQH0), opossum (Q8MJ28), chicken
(Q92072), silkworm (Q5W7N6), Arabidopsis (Q9SEG3), carrot (O48867), corn (Q8LPU6) and rice (A2XMY1). Conserved
amino acids are colored in blue. The red arrows point to the mutations.
30
p.Tyr495Cys, in conserved amino acid in exon 20 of DNMT1
so WHAT?
All mutations are within the targeting-sequence domain of DNMT1.
These mutations cause premature degradation of mutant proteins,
reduced methyltransferase activity and impaired heterochromatin
binding during the G2 cell cycle phase leading to global
hypomethylation and site-specific hypermethylation. Our study shows
that DNMT1 mutations cause the aberrant methylation implicated in
complex pathogenesis. The discovered DNMT1 mutations provide a new
framework for the study of neurodegenerative diseases.
31
NOTE: you are responsible for frameshift,
nonsense and silent mutations even though
we will not cover these terms in class.
32
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
33
Meant to put this question in assignment set 6
A gene may be viewed as a series of binding sites for
RNA and protein
What binds to each of these sites?
34