BIOL 3000 Mutations

Genetic Material “Must haves”

• Effective transfer between generations

• Ability to store vast amounts of information

• Information can be changed/mutable

• Effective replication/high fidelity

• Must be able to keep the mutation and replicate it

What does structure or configuration of a peptide chain confer?

The function of a protein

Where does the structure of a peptide chain come from?

From amino acids and how they interact with each other

If you change the sequence of the DNA or alter the structure of the polypeptide chain, then

You may alter the function of the resulting protein

Types of substitution point mutations

Transition mutant

Transversion mutant

Transition mutant

The exchange of a purine for a purine or a pyrimidine for a pyrimidine. Changing a singular base with a similar/like one.

Ex: ATG → GTG

Purines

Bigger

Adenine and Guanine

Pyrimidines

Smaller

Cytosine and Thymine

Transversion mutant

The exchange of nucleotides outside of a nucleotide family; the exchange of a purine for a pyrimidine.

Ex: ATG → T/CTG

Which happens more often: transitions or transversions? Why?

Transitions because the structure stays the same. Cells do not like using energy, so going from a base to a similar one takes less energy.

Types of insertions/deletions point mutations

Silent

Missense

Nonsense

Frameshift mutants

In-frame mutants

Silent mutations

The changing of one codon to a synonymous codon causing no change in the amino acid sequence of the protein. There is no change in gene expression.

“Degeneracy of Genetic Code”

Which nucleotide do silent mutations usually take place?

Generally, at the third nucleotide of a codon which causes no change in the amino acid coded for.

“A change in genotype causes no change in phenotype”

Missense mutation

The changing of one codon to a different codon, resulting in a change in the amino acid sequence of the protein. It does affect the amino acid.

Which nucleotide do missense mutations usually take place?

Generally, at the first nucleotide. If the first position is changed, then it will almost always change the amino acid.

How is the severity of missense mutations measured?

It typically depends on what amino acids are involved and where the missense mutation occurred

Nonsense mutations

The changing of one codon to a “STOP” codon, resulting in the premature stoppage of translation. It completely changes what the polypeptide is. The effect of the mutation depends on where it is. It makes the ribosome less sensitive to premature STOP codons.

Ex: Cystic fibrosis, Duchenne Muscular Dystrophy (DMD)

Frameshift mutation

The gain or loss of a nucleotide (or nucleotides) that result in the change in the reading frame of the codon. There can be a gain or loss of multiple. This can shift the reading frame, changing everything after the mutation.

It can result in a STOP codon, which would make it a nonsense mutation

Ex: Crohn’s Disease

In-frame mutation

The gain or loss of a nucleotide or trinucleotide set that does not change the reading frame of the codon.

This is a missense mutation.

Types of functional mutants of point mutations

Gain/loss of function mutants

Lethal mutants

Loss-of-Function Mutation

Results in a gene product with little or no functionality, amorphic. Most of the time, these phenotypes are recessive. There is a protein made, but the function is lost.

Gain-of-Function Mutation

Results in a gene product that has gained a new and abnormal function, neomorphic. These mutations are typically dominant.

Amorphic

The complete loss of a gene function

Neomorphic

A new or different function from normal

Lethal mutation

Mutation that leads to the death of the organism carrying the mutation. This is pretty bad, and can be any kind of mutation

Mutation in somatic cells

It can range from mild to severe, but is not passed to the next generation. Most of the time, induced mutations. They may pass down the “tendency” to have something.

Mutation in germ cells

Typically more severe manifestation because they can be passed along to offspring. It is something that can stay in the population.

Ex: breast cancer, hemophilia

Spontaneous mechanism of mutations

Replication errors

Chemical changes

Induced mechanisms of mutations

Environmental factors

Chemical interactions

Spontaneous mutations

Any mutation where no artificial factor or external regulator causes the mutation. Typically happens in replication errors.

Replication errors

Every time a cell divides; it must make an exact copy of 3 billion nucleotides to pass to daughter cells. 6 billion base pairs per diploid cell. The mutation rate of 1 per 100,000 bases.

120,000 mistakes EVERY time a cell divides.

Results in the Non-Watson-and-Crick base pairing

Non-Watson-and-Crick base pairing

AKA “wobble”

Non-complimentary bases can pair due to the flexibility of DNA double helix which can accommodate slightly misshaped pairings.

Slipped Strand Mis-pairing

Involves the denaturation and displacement of DNA strands that results in mispairing of complimentary bases.

Chemical changes

Mutations caused by normal chemical reactions that occur in the cell

Depurination

A chemical reaction in which a β-N-glycosidic bond is cleaved by hydrolysis causing the release of an Adenine or Guanine from a DNA strand. Losing a purine.

Still having a backbone, just no base.

Deamination

The hydrolytic removal of an amine group from a nucleotide releasing ammonia and converting the nucleotide to another nucleotide. Losing an amine group. This is the most common single nucleotide mutation in DNA.

Types of induced mechanisms of mutations

Chemical interactions

Environmental factors

Chemical interactions

5-Bromouracil

5-BrdU

5-Bromouracil

A base analog or antimetabolite of uracil that can replace thymine in a strand of DNA

5-BrdU

The deoxyribose form

Used to study cancer cell proliferation as it is neither radioactive nor toxic to the cell

Environmental factors

Free radicals

UV light

Free radicals

Very unstable and quick reacting molecule that “steals” electrons from nearby stable molecules. They can be missing any number of electrons

UV Light

Causes pyrimidine dimers by the formation of covalent linkages localized on cysteine double bonds

Chromosome duplication

The duplication of a region of DNA that contains a gene

Causes of chromosome duplication

Ectopic recombination

Chromosome deletions

Small: less likely to be deletions

Medium: responsible for a number of genetic diseases

Large: often fatal

Transposable elements (TEs)

AKA “jumping genes” or transposons, mobile sequences of DNA that move (or jump) from one location in the genome to another that often generate some type of mutation when they move from one location to another on a chromosome and in evolutionary terms

It can increase the size of the genome

Might carry out some biological function, most likely regulatory one.

Thought to be junk DNA but now believed to make up about 40% of human genome

What was used to observe transposons?

Corn

Ds

Dissociator (breakage of DNA), this is the actual transposon

It can move multiple different times

Ac

Activator (causes the breakage of DNA) and impacts the expression of the Ds

Class 1 TEs

Requires a reverse transcriptase (the transcription of RNA into DNA) in order to transpose

Retrotransposons

REtrotransposons = REquire reverse transcriptase

Class 2 TEs

Do not require reverse transcriptase in order to transpose

DNA transposons

Mechanism of Class 2 TEs

Autonomous Class 2 TEs encode transposase, DNA transposons

Moves throughout the genome in a “cut and paste” mechanism

Less than 2% of human genome composed of DNA transposons

Uses Terminal Inverted Repeats (TIR), Flanking Direct Repeats (DR)

Terminal Inverted Repeats (TIR)

Inverted compliments of 9-40 base pairs located at both ends of the transposable element. It is recognized by the transposase

Flanking Direct Repeats (DR)

Not a direct part of the transposable element but plays a role in inserting the transposable element back in the genome. It provides a marker for the excision site once a TE has moved. It is part of the DNA and dictates where the TE goes.

How does transposition work?

Before: Goes through transcription and translation, then it is recognized

1. The TE is removed from the DNA sequence by transposase

2. TE/Transposase

3. Staggered cuts are made in the target DNA by transposase, makes overhanging ends

4. The TE inserts itself into another location in the DNA

5. Staggered cuts leave short, ssDNA pieces

6. Replication of the ssDNA creates the flanking DR

Mechanism of Class 1 TEs

Functions through the action of RNA intermediates

Does NOT encode Transposase; encodes reverse transcriptase

Produces RNA transcripts and relies on reverse transcriptase to reverse transcribe the RNA into DNA sequences prior to insertion into target DNA

Moves through the genome in a “copy and paste” mechanism

Has Long Terminal Repeat transposons (LTR) and Non-Long Terminal Repeat transposons (NLTR)

Long Terminal Repeat (LTR) Transposons

Characterized by the presence of LTR on each end of the TE. Each end has satellites of repeated DNA and can be autonomous

Not in human genome

Non-Long Terminal Repeat (NLTR) Transposons

LTR is not present and is the ONLY active class of transposons in humans

Autonomous and non-autonomous

Autonomous (Long Interspersed Elements- LINEs)

Capable of moving on their own as they make their own Reverse Transcriptase required to move

DNA sequences that range in length from a few hundred to as many as 9,000 base pairs; they’re very long because they actually have the gene for reverse transcriptase and can make their own proteins.

Functional L1 elements are about 6,500 bps in length and encode proteins, including an endonuclease that cuts DNA and a reverse transcriptase that makes a DNA copy of an RNA transcript

Most L1 elements are not functional, they may play a role in regulating the efficiency of transcription of the gene in which they reside

Non-autonomous (Short Interspersed Elements- SINEs)

Must “borrow” reverse transcriptase from another element in order to move. Cannot make reverse transcriptase, so they use reverse transcriptase that comes from LINEs.

Short DNA sequences (100-400 bps) that represent reverse-transcribed RNA molecules originally transcribed by RNA pol II

Represents some 10% of total DNA

Most abundant SINEs are the Alu elements consisting of a sequence that contains a site that is recognized by the restriction enzyme Alu1

Alu elements

A transposon; it codes for all the proteins and enzymes needed to move a transposon

5’-AG/CT-3’ or 3’-TC/GA-5’

Occurring in the introns of genes which can be spliced into mature mRNA creating a new exon, which will be transcribed into a new protein product (way of possibly creating new proteins)

Alu1 Mechanism

If the Alu1 happens to sit down in the middle of the intron, then it is spliced along with the exons. Then the Alu1 will become an exon, if this will be able to code for a protein, then it is a new protein product. Increasing the genetic diversity.

How does retrotransposition work?

1. Retrotransposon sequence is transcribed to RNA.. mRNA

2. Undergoes reverse transcriptase to produce dsDNA, winded up with double stranded molecule

3. Staggered cuts are made in the target DNA

4. Retrotransposon integrates into the new site in the host DNA

   1. Replication fills in the gaps at the site of insertion creating FDR, unbalanced. Copied and added DNA

What do Transposable Elements do?

For the most part, it just depends on where it lands.

In non coding regions, nothing much

In genes, it can result in a mutation; however, keep in mind that not all mutations are deleterious

What leads to genetic diversity?

Mutations