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Methods used to study mutations

Gross chromosomal changes- deletions, insertions, inversions, translocations

Cytology- microscopy- karyotype

Small mutationsSmall deletions, insertions and point mutations

Recombinant DNA technologies

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Frameshift mutations

A single base-pair deletion or insertion results in a change in the reading frame

AUG UUU AGC UUU AGC UUU AGC WT

Met Phe Ser Phe Ser Phe Ser

Delete C

AUG UUU AGU UUA GCU UUA GC

Met Phe Ser Leu Ala Leu

Insert C

AUG UUU AGC CUU UAG CUU UAG C

Met Phe Ser Leu STOP

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Frameshift mutations- Deletion

A single base-pair deletion or insertion results in a change in the reading frame

AUG UUU AGC UUU AGC UUU AGCMet Phe Ser Phe Ser Phe Ser

Delete CAUG UUU AGU UUA GCU UUA GCMet Phe Ser Leu Ala Leu

Delete GCAUG UUU AUU UAG CUU UAG CMet Phe Ile Stp

Delete AGCAUG UUU UUU AGC UUU AGCMet Phe Phe Ser Phe Ser

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Frameshift mutations-Insertion

A single base-pair deletion or insertion results in a change in the reading frame

AUG UUU AGC UUU AGC UUU AGCMet Phe Ser Phe Ser Phe Ser

Insert CAUG UUU AGC CUU UAG CUU UAG CMet Phe Ser Leu STOP

Insert CCAUG UUU AGC CCU UUA GCU UUA GCMet Phe Ser Pro Leu Ala Leu

Insert CCCAUG UUU AGC CCC UUU AGC UUU AGCMet Phe Ser Pro Phe Ser Phe Ser

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Missense mutations

Missense mutations alters ONE codon so that it encodes a different amino acid

UUU UUU UGC UUU UUU WTPhe Phe Cys Phe Phe

UUU UUU UGG UUU UUU mutPhe Phe Trp Phe Phe

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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 un structured tails

Silent mutations

Mutations in non-coding regions

Nonsense mutations

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Silent Mutations

Silent mutations do not alter the amino acid sequence!The Genetic code is degenerate!

AUG UUU AGC UUU AGC UUU AGC WTMet Phe Ser Phe Ser Phe Ser

AUG UUC AGC UUU AGC UUU AGC MutMet Phe Ser Phe Ser Phe Ser

Mutations that occur in introns are also silent

Mutations that occur in non-genic regions are often silent

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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 proteinsORIncreased 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

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Lactose intolerance in humans

Lactose========>Glucose + GalactoseLactase

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 inheritence of different alleles of the lactase gene.

Lactose intolerance is the result of being homozygous for the recessive lactase (WT) alleleBeing homozygous or heterozygous for the mutant allele allows lactase expression in adults when normally lactase expression is turned off.

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•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

C/C

T/T

CCCCAGGC

There are no mutations in the coding region of the lactase gene.

A mutation is observed in the enhancer -13910 bp upstream of the gene in an AP2 consensus sequence.

lactose tolerance

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Mutations in splicing of RNA

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Nonsense mutations

Nonsense mutations alter one codon so that it now encodes for a STOP codon

UUU UUU UGC UUU UUUPhe Phe Cys Phe Phe

UUU UUU UGA UUU UUUPhe Phe STOP

Nonsense mutations insert a stop codon which results in premature termination

Truncated polypeptide usually results in loss of function for polypeptide

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There are NO tRNAs in 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

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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.

Point mutation occurs in the anticodon loop OF THE tRNAThis allows this tRNA to base pair with a stop codon and ?

AUG

Trp

---UAC---UAGAUC

Trp

---UAG---UAG

DNA

Gene encoding tRNATRP

Normal tRNA Mutant tRNA

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--- UUU UUU UAG UUU UUU -------- Phe Phe STOP

--- Phe Phe Trp Phe Phe ---->

A mutant protein that is larger than normal will be synthesized!!

5’--- UUU UUU UAG UUU UUU -----3’AUC

Trp

AAA

MetAla

PhePhe

Trp-tRNA has mutationIn anticodonThis allows it to pairwith a stop codon

Nonsense suppressor

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Nonsense and Nonsense suppressor

--- UUU UUU UAG UUU UUU -------- Phe Phe STOP

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.

AUC

Trp

---UAG---

5’--- UUU UUU UAG UUU UUU -----3’AUC

Trp

AAA

MetAla

PhePhe

AAA

Phe

AAA

Phe

--- UUU UUU CAG UUU UUU -------- Phe Phe Gln Phe Phe ---

Nonsense mutation

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Recombinant DNA technology

When genes are mutated - proteins are mutated- DISEASE STATES OCCUR

Sickle cell Anemia

Globin2 alpha globin chains2 beta globin chains Mol wt 16100 daltons xfour = 64650 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 necessaryTake advantage of enzymes and reactions that naturally occur in bacteria

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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!

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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 phenotypeIt usually provides No Information about how the protein generates the phenotype

For p53We 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 informationIs the location in the cell affected

How does it influence the behavior of the cell during division

Organism phenotype