1. Lect10: Mutations
6th Ed: Ch 8-1, 7-1, 7-2, 8-5

2. What is a gene? Prokaryotic Genes PROMOTER 3’ 5’ 3’ (-35) (-10)
antisense
---TTGACAT------TATAAT AT-/-AGGAGGT-/-ATG CCC CTT TTG TGA
---AACTGTA------ATATTA TA-/-TCCTCCA-/-TAC GGG GAA AAC ATT
sense 3’
(-35)
(-10)
RIBOSOME
BINDING
SITE 5’ 5’ 3’
U-/-AGGAGGU-/-AUG CCC CUU UUG UGA
Met Pro leu leu stp
When all of these rules are satisfied then A segment of DNA will generate an RNA which will then be read by a ribosome and be translated into a protein.

3. Reading the genetic code
A T G
T T T
A A A
T A G
C C C 5’ 3’
C A T
A A A
T T T
C T A
G G G 5’ 3’
A T G
T T T
A A A
T A G
C C C 5’ 3’ 5’ 3’
A U G U U U A A A U A G C C C 5’ 3’
C A T
A A A
T T T
C T A
G G G
A U G
U U U
A A A
U A G
C C C 5’ 3’
U A C
Met
A A A
Phe
U U U
Lys
S T P

4. No Gaps
A U G
U U U
A A A
U A G
C C C 5’ 3’
U A C
Met
A A A
Phe
U U U
Lys
S T P
A U G
U U U
A A A
U A G
C C C 5’ 3’
U A C
Met
A A U
Asn
U U A
Leu

5. No overlaps
A U G
A A A
C C C
U A G 5’ 3’
U A C
Met
U U U
Lys
G G G
Pro
S T P
A U G
A A A
C C C
U A G 5’ 3’
U A C
Met
U U U
Lys
U G G
Trp

6. The GENETIC CODE The code is a three letter code. Second letter U C A
UUU
UUC
UUA
UUG
CUU
CUC
CUA
CUG
AUU
AUC
AUA
AUG
GUU
GUC
GUA
GUG
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
UAU
UAC
UAA
UAG
CAU
CAC
CAA
CAG
AAU
AAC
AAA
AAG
GAU
GAC
GAA
GAG
UGU
UGC
UGA
UGG
CGU
CGC
CGA
CGG
AGU
AGC
AGA
AGG
GGU
GGC
GGA
GGG U C A G
Phe
Tyr
Cys U
Ser
STOP
STOP
Leu
Trp
His C
Arg
Pro
Leu
Gln
First letter
Third letter
Asn
Ser
Ile A
Thr
Lys
Arg
Met
Asp G
Val
Ala
Gly
Glu

7. The Genetic Code Properties of the Genetic code:
1- The code is written in a linear form using the nucleotides
that comprise the mRNA
2- The code is a triplet: THREE nucleotides specify
ONE amino acid
3- The code is degenerate: more than one triplet specifies
a given amino acid
4- The code is unambiguous: each triplet specifies only
5- The code contains stop signs- There are three different stops
6- The code is comma less
7- The code is non-overlapping

8. xxxxxx

9. Mutations
Most mutations are harmful in their effects; only rarely are mutations beneficial.
A gene with one wild-type allele is monomorphic; a gene with two or more wild-type alleles is polymorphic.
The vast majority of mutant traits are determined by alleles of more than one gene. This means that most traits are multifactorial traits.
A Heterogeneous Trait is one that may be caused by mutations in more than one gene.
Human deafness is an example of a heterogeneous trait: Mutations in any of at least 50 genes lead to deafness.
An important class of mutations are conditional mutations- (Environment affects Phenotype). Conditional mutations are those that express their associated phenotype only under some conditions (restrictive conditions) and not others (permissive conditions).
Conditional lethal mutations are common.
Temperature-sensitive conditional mutations are invaluable in genetic research.

10. Generation of mutations
Spontaneous mutations
Replication induced mutations of DNA
Usually base substitutions (Most errors are corrected)
Meiosis- segregation defects or defects during crossing over can induce mutations
Small additions and deletions AND Large changes as well
Environment induced changes
Exposure to physical mutagens - Radioactivity or chemicals
Depurination (removal of A or G)
Repair results in random substitution during replication
Deamination (removal of amino group of base) (nitrous acid)
Cytosine--uracil--bp adenine--replication--
Oxidation (oxoG)
guanine--oxoguanine--bp adenine--replication --
Base analog incorporation during replication BU-T
Intercalating agents

11. Mutation rate There are approximately 1013 cells in the human body
Each cell receives ,000 DNA lesions per day (Lindahl and Barnes 2000). Almost all are repaired!!
Most pervasive mutagen is UV. 100,000 lesions per exposed cell per hour (Jackson and Bartek 2009).
Ionizing agents (X-rays/g-rays) are most toxic because they generate double strand breaks (Ward 1988).
Chromosome instability (gain or loss of entire segments) is frequent - 40% of imbalances are entire arm imbalance while 45% are terminal segment imbalance (double strand break, nondysjunction etc)
Sequencing 179 humans as part of the 1000 genome project:
On average, each person is found to carry approximately 250 to 300 loss-of-function variants in genes of which 50 are in genes previously implicated in genetic disorders.
Each individual on average has:
1.3 million short indels (1-10,000 bp) and 20,000 large sequence variations (>10,000 bp).
Variation detected by the project is not evenly distributed across the genome: certain regions, containing repetitive sequences (sub-telomeres etc), show high rates of indels.

12. Sequencing the whole genomes of a family (2010 Science 328 636).
98 crossovers in maternal genome
57 crossovers in paternal genome
Mutation rate is 1x10-8 per position per haploid genome
(human genome is 3x109 bp)
It was calculated that there are ~70 new alterations in each diploid human genome
Some sites such as CpG sites mutate at a rate 11 times higher than other sites
Exome sequencing of 2440 individuals (Science )
Each person has ~100 loss of function mutations (~35 nonsense).
20 loss of function mutations are homozygous
Some alterations in sequence concentrate in specific geographic populations.
Rare changes are population specific and their frequencies vary for each geographic population

13. Methods used to study mutations
Gross chromosomal changes-
deletions, insertions, inversions, translocations
Cytology- microscopy- karyotype
Small mutations
Small deletions, insertions and point mutations
Recombinant DNA technologies

14. This means that most traits are multifactorial.
Most mutations are harmful in their effects; only rarely are mutations beneficial.
A gene with one wild-type allele is monomorphic; a gene with two or more wild-type alleles is polymorphic.
The vast majority of traits are determined by alleles of more than one gene.
This means that most traits are multifactorial.
A Heterogeneous Trait is One That May be caused by mutations in more than one gene.
Human deafness is an example of a heterogeneous trait: mutations in any of at least 50 genes lead to deafness.
(How can one tell if two deaf individuals carry mutations in the same gene or mutations in different genes?)
An important class of mutations are conditional mutations.
In these mutants the Environment affects Phenotype.
Conditional mutations are those that express their associated phenotype only under some conditions (restrictive conditions) and not others (permissive conditions).
Conditional lethal mutations are common.
Temperature-sensitive conditional mutations are invaluable in genetic research.

15. Missense mutations UUU UUU UGC UUU UUU WT Phe Phe Cys Phe Phe
Missense mutations alters ONE codon so that it encodes a different amino acid
UUU UUU UGC UUU UUU WT
Phe Phe Cys Phe Phe
UUU UUU UGG UUU UUU mut
Phe Phe Trp Phe Phe

16. Consequences of Missense Mutations
Missense mutations alter one of the many amino acids
that make a protein
Its consequences depend on which amino acid is altered
Conservative mutations: K to R
Nonconservative mutations: K to E
Surface Vs buried
Mutations in globular domains Vs unstructured tails
Silent mutations
Mutations in non-coding regions
Nonsense mutations

17. NNN UGG CGA UGG CGA UGG CGA UGG CGA NNN
Frameshift mutations
A single base-pair deletion or insertion results in a change
in the reading frame
NNN UGG CGA UGG CGA UGG CGA UGG CGA NNN
Trp Arg Trp Arg Trp Arg Trp Arg
Delete A
NNN UGG CGU GGC GAU GGC GAU GGC GAN NN
Trp Arg Gly Asp Gly Asp Gly
Insert A
NNN UGG CGA AUG GCG AUG GCG AUG GCG ANNN
Trp Arg Met Ala Met Ala Met Ala

18. NNN UGGCGAUGGCGAUGGCGAUGGCGA NNN ... TrpArgTrpArgTRpArgTrpArg …
Frameshift mutations
A single base-pair deletion or insertion results in a change
in the reading frame
NNN UGGCGAUGGCGAUGGCGAUGGCGA NNN
... TrpArgTrpArgTRpArgTrpArg …
.... GlyAspGlyAspGlyAspGlyAsp
..... AlaMetAlaMetAlaMetAla
Delete A
UGG CGU GGC GAU GGC GAU GGC GANN
Trp Arg Gly Asp Gly Asp Gly
Insert A
UGG ACG AUG GCG AUG GCG AUG GCG ANN
Trp Thr Met Ala Met Ala Met Ala

19. Frameshift mutations- Deletion
Base-pair deletion or insertion results in a change
in the reading frame
AUG UUU AGC UUU AGC UUU AGC
Met Phe Ser Phe Ser Phe Ser
Delete C
AUG UUU AGU UUA GCU UUA GC
Met Phe Ser Leu Ala Leu
Delete GC
AUG UUU AUU UAG CUU UAG C
Met Phe Ile Stp
Delete AGC
AUG UUU UUU AGC UUU AGC
Met Phe Phe Ser Phe Ser

20. Frameshift mutations-Insertion
Base-pair deletion or insertion results in a change
in the reading frame
AUG UUU AGC UUU AGC UUU AGC
Met Phe Ser Phe Ser Phe Ser
Insert C
AUG UUU AGC CUU UAG CUU UAG C
Met Phe Ser Leu STOP
Insert CC
AUG UUU AGC CCU UUA GCU UUA GC
Met Phe Ser Pro Leu Ala Leu
Insert CCC
AUG UUU AGC CCC UUU AGC UUU AGC
Met Phe Ser Pro Phe Ser Phe Ser

21. AUG UUU AGC UUU AGC UUU AGC WT Met Phe Ser Phe Ser Phe Ser
Silent Mutations
Silent mutations do not alter the amino acid sequence!
The Genetic code is degenerate!
AUG UUU AGC UUU AGC UUU AGC WT
Met Phe Ser Phe Ser Phe Ser
AUG UUC AGC UUU AGC UUU AGC Mut
Mutations that occur within introns are often silent but mutations at splicing junctions have effects
Mutations that occur in non-genic regions are often silent except when they occur in regulatory sequences

22. Mutations in non-protein coding regions
Mutations in the promoter, splicing junction or ribosome binding site are also mutagenic
Reduced expression of mRNA might result in reduced levels
of proteins OR
Increased expression of mRNA might result in increased levels of protein
Mutations in splicing junctions may also be mutagenic improperly spliced mRNA will result in the intron being translated
Mutations in tRNA or aminoacyl-tRNA synthase are mutagenic

23. Lactose intolerance in humans
Lactose========>Glucose + Galactose
Lactase
Human milk is 7% lactose. Lactose is not absorbed through the wall of the digestive tract.
In human infants, lactase is secreted in intestine which breaks the lactose into easily absorbed Glucose and Galactose.
Production of the lactase enzyme declines in adults.
The unabsorbed lactose creates cramps, diarrhea, and nausea.
In some humans, lactase continues to be produced throughout adulthood. These individuals are called lactose absorbers (LA).
Adult lactose absorption is inherited as an autosomal dominant trait.
Lactose persistence and non-persistence reflect inheritance of different alleles of the lactase gene.
Lactose intolerance is the result of being homozygous for the recessive lactase (WT) allele
Being homozygous or heterozygous for the mutant allele allows lactase expression in adults when normally lactase expression is turned off.

24. Lactose tolerance
There are no mutations in the coding region of the lactase gene.
A mutation is observed in the enhancer bp upstream of the gene in an AP2 consensus sequence.
CCCCAGGC
the polymorphism modifies a transcription factor binding site (AP2)
AP2 acts as a repressor but in the mutant it cannot bind and cannot repress the gene- so adults keep producing lactase
CCCCAGGC
TCCCAGGC

25. Synuclein Gene and Parkinson
At one nucleotide in a non-protein-coding region of SNCA, the gene that encodes α-synuclein, the presence of the base adenine (A) is protective against Parkinson's disease, whereas the presence of another, guanine (G), confers increased risk. Soldner et al.3 report that this region regulates SNCA expression levels. If the two copies of the chromosome in a human cell each contain a different base at this site, gene expression is significantly higher from the risk-variant chromosome, owing to a reduction in the attachment of DNA-binding proteins that inhibit transcription. Using CRISPR–Cas9 gene-editing technology to remove the G and replace it with A reduces SNCA expression.

26. Mutations in splicing of RNA

27. UUU UUU UGC UUU UUU Phe Phe Cys Phe Phe UUU UUU UGA UUU UUU
Nonsense mutations
Nonsense mutations alter one codon so that it now encodes
for a STOP codon
UUU UUU UGC UUU UUU
Phe Phe Cys Phe Phe
UUU UUU UGA UUU UUU
Phe Phe STOP
Nonsense mutations insert a stop codon which results in
premature termination
Truncated polypeptide usually results in loss of function
for polypeptide

28. A U G U A C A A A U A G C C C 5’ 3’ U A C Met A U G Trp U U U Lys
There are NO tRNAs in wildtype cells with anti-codons that recognize STOP codons in mRNA
What happens if there is a mutation in the anti-codon loop of a specific tRNA Gene that allows a tRNA to recognize a stop codon
A U G
U A C
A A A
U A G
C C C 5’ 3’
U A C
Met
A U G
Trp
U U U
Lys
S T P

29. Nonsense suppressor mutations!
These are the result of a mutation in the anti-codon loop of
a specific tRNA Gene
It allows the tRNA to recognize a nonsense codon and base
pair with it.
DNA
Gene encoding tRNATRP
Point mutation occurs in the anticodon loop OF THE tRNA
This allows this tRNA to base pair with a stop codon and ?
AUG
Trp
AUG
Trp
AUC
Trp
5---UAC---UAG---UAA3’
5--UAC---UAG---UAA3’
Normal tRNA
Mutant tRNA

30. Nonsense suppressor --- UUU UUU UAG UUU UUU ----- --- Phe Phe STP
Trp-tRNA has mutation in anticodon. This allows it to pair with a stop codon
5’--- UUU UUU UAG UUU UUU UAA-----3’
AUC
Trp
AAA
Met
Ala
Phe
--- Phe Phe Trp Phe Phe |
A mutant protein that is larger than normal will be synthesized!!

31. Nonsense and Nonsense suppressor
--- UUU UUU CAG UUU UUU -----
--- Phe Phe Gln Phe Phe ---
Nonsense mutation
--- UUU UUU UAG UUU UUU -----
--- Phe Phe STOP
AUC
Trp
---UAG---
What will happen if an individual carries both a nonsense mutation in a gene and a nonsense suppressor mutation in the anticodon loop of one of the trp-tRNA genes.
5’--- UUU UUU UAG UUU UUU ’
AUC
Trp
AAA
Met
Ala
Phe
Phe Phe Trp Phe Phe

33. Methods used to study mutations
Gross chromosomal changes-
deletions, insertions, inversions, translocations
Cytology- microscopy- karyotype
Small mutations
Small deletions, insertions and point mutations
Recombinant DNA technologies

34. Recombinant DNA technology
When genes are mutated - proteins are mutated-
DISEASE STATES OCCUR
Sickle cell Anemia
Globin
2 alpha globin chains
2 beta globin chains
Mol wt daltons xfour = daltons
Single point mutation in beta-globin
Converts Glu to Val at position 6
Need to know mutation
Need to look at genes of individuals
Genes lie buried in 6billion base pairs of DNA
(46 chromosomes).
Molecular analyses necessary
Take advantage of enzymes and reactions that naturally
occur in bacteria

35. Why all the Hoopla? Why all the excitement over recombinant DNA?
It provides a set of techniques that allows us to study
biological processes at the level of individual proteins
in individuals!
It plays an essential role in understanding the genetic basis
of cancer in humans
Recently found that mutations in a single gene called p53
are the most common Genetic lesion in cancers.
More than 50% of cancers contain a mutation in p53
Cells with mutant p53
Chromosomes fragment
Abnormal number of chromosomes
Abnormal cell proliferation!

36. Alkaptonuria
Degenerative disease. Darkening of connective tissue, arthritis
Darkening of urine
Garrod characterized the disorder- using Mendels rules- Autosomal recessive. Affected individuals had normal parents and normal offspring.
1909 Garrod termed the defect- inborn error (genetic) of metabolism. Homogentisic acid is secreted in urine of these patients. This is an aromatic compound and so Garrod suggested that it was an intermediate that was accumulating in mutant individuals and was caused by lack of enzyme that splits aromatic rings of amino acids.
1958 La Du showed that accumulation of homogentistic acid is due to absence of enzyme in liver extracts
Garrods results and his explanation were ignored
1994 Seidman mapped gene to chromosome 3 in human
1996 Gene cloned
1996 Mutant identified P230S &V300G
2000 Enzyme activity shown to be principally in liver and kidneys

37. p53 To understand the complete biological role of p53 protein
and its mutant phenotype we need to study the gene at
multiple levels:
Genetics- mutant gene- mutant phenotype
Now what?
Genetics will relate specific mutation to specific phenotype
It usually provides No Information about how the protein
generates the phenotype
For p53
We would like to know
The nucleotide sequence of the gene and the mutation that leads to cancer
When and in which cells the gene is normally expressed
(in which cells is it transcribed)
At the protein level--Amino acid sequence
Three-dimensional structure
Interactions with other proteins
Cellular information
Is the location in the cell affected
How does it influence the behavior of the cell during division
Organism phenotype