D1.3 MUTATIONS & GENE EDITION

Define gene mutations and the type of mutations that can occur

DEFINITION: Gene mutations are structural changes or alterations in the gene of the DNA sequence

TYPES:

1. Single base substitution: a single nucleotide/base is replaced

   • Single-nucleotide polymorphisms (SNPs) is when one nucleotide is replaced by another nucleotide in DNA

   • Ex: Sickle cell anaemia in the mutation in the HBB gene (GAG to GTG)

2. Insertion: the addition of one or more nucleotides into a gene

   Ex: Huntington’s disease (CAG repeasts are inserted into the hTT gene

3. Deletion: Loss of one more more nucleotides from a gene

4. Frameshift: Due to insertion or deletion mutations that are not in a multiple of 3. Every codon after the mutation is read incorrectly

Outline the consequences of base substitution mutation, including an example

1. Genes are a sequence of DNA bases that determines the amino acid sequence of a protein

2. Changing a single base in DNA can cause the mRNA to code for a different amino acid and a different protein to be made during translation

3. Mutation in the HBB gene can change the protein structure of haemoglobin

Discuss why base substitution mutations may not be harmful

1. A change in the base sequence/codon might not change the amino acid

2. Multiple codons code for each amino acid

3. The genetic code is degenerate

4. Mutagens (radiation and mutagenic chemicals) increase DNA mutations

5. DNA proofreading and repair mechanisms by DNA polymerase (I and II) may fail

Outline the cause and consequences of sickle cell anaemia

CAUSE:

1. DNA nucleotides GAG are changed into GTG

2. The mutation is a base substitution mutation occurring in the 6th codon of the sense strand

3. The Hb^A allele of the gene is mutated forming a new allel/version of the gene called Hb^S

4. Mutation changes the mRNA strand produced during transcription, in which GAG is converted into GUG in mRNA

5. A different tRNA binds to the mRNA codon, so glutamic acid is changed to valine — a different amino acid is inserted into the polypeptide, changing the amino acid sequence

CONSEQUENCE:

1. Beta-globin subunit (polypeptide) of haemoglobin crystallises → Red blood cells become sickle shaped

2. Haemoglobin has vital role: when sickle cell haemoglobin releases oxygen, it becomes less soluble and crystallises

3. Blocks blood flow to peripheral tissues, impairs blood flow in capillaries, decreases transport of oxygen to tissues → anaemia: blood flow and tiredness

4. Red blood cells live for 8 days, normal blood cells live for 80 days

Outline the consequences of insertion and deletions with two examples

1. Insertions and deletions may lead to frameshift mutations if a multiple of 3 nucleotides is not inserted or deleted

2. This is when the reading frame of triplet codons is shifted

3. Insertion (extra base pairs), deletion (base pairs are lost) → all of the AA after the mutation are affected

4. Catastrophic effects of the functionality of the protein

EXAMPLE 1 : Huntington disease

1. On chromosome 4, the HTT gene normally has 10-35 CAG repeats

2. The insertion of CAG repeats leads to 39+ CAG repeats → change in the structure of the huntingtin protein

3. A neurodegenerative disease affects the individual’s mood, ability to talk, and coordination

4. HD is inherited and a dominant allele → inherited from parents

5. Individuals with HD will express it at a later age and receive it from one of their parents

EXAMPLE 2: Delta 32

1. Delta 32 mutation of the CCR5 Gene → deletion mutation

2. Delta 32 is a recessive allele

3. Prevents the expression of the CCR 5 receptor protein used by HIV (to bind) to enter CD-4 cells

4. CCR5 mutation inhibits HIV from entering and infecting cells → lowers infection

5. Mutation provides resistance, lower infection rates

Discuss the causes of gene mutation

1. Mutagens are factors that cause or increase the frequency of mutations

   • forms new alleles of a gene

   • could result in genetic diseases or cancer

   • if it causes cancer, it’s called a carinogen

2. Common mutagens include:

   • high energy/ionising radiation: UV, X-ray, Gamma, beta & alpha particles

   • mutagenic chemicals (mustard gas, cigarette smoke, nitrites in cured meat)

   • Some viruses (papillomavirus)

   • some bacteria (helicobacter pylori)

3. Errors in DNA replication

   • may cause change in protein structure/function

   • DNA polymerase III and I proofreading may fail

Outline the randomness in mutation

1. Mutations can occur randomly anywhere in the base sequence of a genome

2. Cytosine has a relatively high mutation rate compared with the other bases

3. Errors in DNA replication are random:

   • Transversions substitute prymiridnes and purines

   • Transitions substitutes bases/nucleotides of similar shape

4. Evolution: by natural selection is a process that selects favourable alleles generated by random mutation

Discuss the consequences of mutation in germ and somatic cells

GERM CELLS: Reproductive cells — haploid - n, cells that produce sperm and egg

1. Mutations occur in the ovaries/testis/ spermatogonia/oogonia

2. Passed onto the offspring/inherited and affect all of the cells in the offspring

3. Can change the base sequence of the DNA/gene or lead to chromosomal mutations

SOMATIC CELLS: Body cells — all of the cells except reproductive/germ cells, diploid - 2n

1. Mutations occur in body cells

2. Are not inherited and only affect hte individual and the cell/tissue/organ

3. Can lead to the development of cancer

Discuss mutation as a source of genetic variation

The original source of all genetic information is mutation

1. Random mutation creates new alleles

2. Mutations are essential for creating genetic variation → required for evolution through natural selection

3. Mutations in non-coding regions are neutral and don’t effect the phenotype

4. Mutations in coding regions can affect protein structure and be harmful/beneficial/silent

   • Harmful: change protein structure & function (ex huntington’s disease)

   • Beneficial: provide advantageous trait

   • Silent: don't change corresponding amino acid (degenerate genetic code)

Outline gene knockout

1. Investigates the function of the gene by removing/destroying/knockout the function of a particular gene (by deletion)

2. The expression/production of protein is prevented

3. Uses model organisms (non-human organisms)

   • Can do so because of universal genetic code & common biological processes

   • Nematode (organ development and apoptosis), fruit fly (parkinson’s and alzhiemer’s)

   • Zebra fish (transparent tissues for studying circulation and metastatic), Arabidopsis (floral production and environmental stress)

4. Librarires of KO organisms with KO genes allow scientists to understand role of genes and their respective proteins/impacts on biological processes

EX: p53 Gene Knockout (KO)

1. p53 produces a protein that regulates the cell cycle

2. The absence of this gene/p53 KO mice resulted in an increase of tumour development and cancer growth

Outline CRISPR sequences and the enzyme involved in gene editing

CRISPR stands for: Clustered, regular, inter, spaced, palindromic, repeats

1. CRISPR are specific regions of DNA (short, repeated sequences of DNA and spacer DNA) in prokaryotes that are incorporated from a viral attack

2. Singled guide RNA (sgRNA) targets specific genes for modification or deletion

3. sgRNA guides the Cas9 enzyme to make cuts — double strand breaks in DNA

4. Cas9 are endonucleases that target CRISPR spacer DNA to cut the DNA at specific locations with the end sequence NGG (N can be any of the 4 bases) or PAM sequences (proto spacer adjacent motif)

Outline the uses of CRISPR sequences and the enzyme Cas9

USE:

1. Specific genes can be knockout - deleted

2. Genes of interests can be inserted into the host DNA after CRISPR makes a cut in the DNA - addition

3. DNA can be modified/change

GENE THERAPY: Treating genetic disease like sickle cell anaemia

AGRICULTURE: Increase crop yield, nutritional content, disease resistance

DISEASE MODELLING: Use animal models to KO, add or change genes to gain insight into disease progression

GENETIC ENGINEERING: enhance the production of valuable compounds, biofuels enzymes

Outline definition & hypotheses to account for conserved/high conserved sequences in genes

1. Conserved sequences are identical or similar DNA/RNA sequences across a species or a group of species

2. Highly conserved species are identical or similar over long periods of evolution

3. Highly & highly conserved sequences provide evidence for the structure and function of LUCA

4. HYPOTHESIS 1: functional requirement for the gene products: proteins associated with cellular stability, function and reproduction

5. HYPOTHESIS 2: slower rates of mutation:

   • mutation rates are linked to level of gene expression

   • highly transcribed genes have lower rates than expressed genes

   • the templates has lower mutation rates than the sense strand

   • proofreading & repair mechanisms may lower the mutation rates of those section of DNA

6. EXAMPLES: ribosome strcutre, HBA gene/alpha hemoglobin chain, non-coding regions (regulatory DNA & transcription factors)