Gene Mutation Types and Significance

• Gene mutations result in structural changes to genes at the molecular level
• Single-nucleotide polymorphisms (SNPs) come from base substitution mutations
• SNPs may or may not change a single amino acid in a polypeptide due to the degeneracy of the genetic code
• Insertions and deletions can cause polypeptides to lose function completely, through frameshift changes or major alterations
• Trinucleotide repeats of the HTT gene serve as an example of insertions
• The delta 32 mutation of the CCR5 gene serves as an example of deletions
• Gene mutations happen because of mutagens and errors in DNA replication/repair
• Examples of mutagens include chemical mutagens and mutagenic forms of radiation
• Mutations occur randomly throughout a genome's base sequences, though some bases are more prone to mutation
• There's no known natural way to deliberately alter a specific base to change a trait
• Mutations in germ cells can be inherited, while those in somatic cells can lead to cancer
• Gene mutation is the source of all genetic variation
• Most mutations are harmful/neutral to an organism, they are essential for evolution through natural selection in the long run

Gene mutations as structural changes

• Gene mutation involves a change in the DNA sequence, altering the amino acid sequence that is transcribed and translated
• Gene mutations are mainly categorized as base substitutions, deletions, and insertions
• Chromosome mutations affect entire chromosomes
• Chromosome mutations are more serious and often result in lethal consequences
• Point mutation involves the substitution of a single base
• Substitution involves one base being replaced by another
• Effect of substitution may be minimal if it is the third base of the codon
• A more serious effect results from the mutation impacting the first or second base of the codon, as it alters the amino acid
• The effect depends on whether the mutation is in the sense or antisense strand
• Sense/antisense strand influences dependency on the template
• Deletion or Insertion are frameshift mutations, often harmful since they shift the reading frame, changing the amino acid sequence
• The resulting protein is not usually functional

Consequences

• The consequences of base substitution mutations depend on the location in the DNA
• A point mutation in the HOX gene in fruit flies (Drosophila melanogaster) can cause legs to grow on the forehead instead of antennae
• The human genome contains mostly non-coding DNA (98%) that is not transcribed/translated, with only 2% coding for proteins
• Base substitutions in non-coding regions between genes are less likely to have an effect

Types of base substitution mutations

• Silent mutations involve a change to a triplet in DNA, but have no effect on the mRNA codon
• Silent mutations do not alter the protein sequence
• Missense mutations replace an amino acid, such as replacing proline with threonine
• Missense mutations alter the polypeptide structure and function, potentially leading to conditions like sickle cell anemia
• Nonsense mutations result in a stop codon replacing an amino acid
• Nonsense mutations shorten the polypeptide chain, rendering it non-functional

Consequences of insertions and deletions

• Frameshift mutations from insertions or deletions alter the reading frame, often causing the polypeptide to completely lose function
• Frameshift mutations can occur by spontaneous (strand slippage) or induced (mutagens) processes

Case studies of frameshift mutations

• Deletion of the CCR5 receptor (Δ32 mutation) prevents the protein from being produced, making the person less likely to contract HIV since the virus cannot enter cells
• Huntington's disease results from repeating trinucleotide repeats (CAG) in the HTT gene
• Huntington's is a neurodegenerative genetic disorder linked to chromosome 4
• Individuals with the Huntington's mutation have more than 39 CAG repeats, leading to early-onset dementia
• The mutation is dominant but expresses itself after reproductive age

Data Regarding BRCA1 mutations

• BRCA1 is a tumor suppressor gene with over 500 variants that increase the risk of various cancers
• Frameshift mutations are the most common type of BRCA1 mutation found in breast cancer patients
• Mutations are random changes to the base sequence
• There are far more possible missense mutations than nonsense mutations
• Nonsense mutations have stop codons, with less possibilities, but are more common in patients
• Ashkenazi Jewish groups have a smaller gene pool and founder effect, African American have a larger gene pool

Cause of gene mutations

• Random changes to the base sequence caused by DNA repair/replication errors and mutagens cause gene mutations
• Mutagenic forms of radiation and chemical compounds can also be carcinogens that result in uncontrolled cell division and tumor formation
• UV radiation and smoking are common mutagens that frequently lead to skin and lung cancer, respectively
• Other mutagens include asbestos, nitrate/nitrite preservatives, smoked foods, and mustard gas
• Radiation increases the mutation rate if it is sufficiently high in energy or chemical changes to DNA
• Ionizing radiation (X-rays, gamma rays) have shorter wavelengths and trigger formation of damaging ions and radicals
• Non-ionizing radiation (UV) is known as less dangerous, but is absorbed by nitrogen bases, causing mismatch of bases (thymine dimers) and malfunction of oncogenes

Randomness in genetic mutationsD1.3.5

• Mutations can occur anywhere in the base sequences of a genome, some bases have a higher probability of mutating
• No natural deliberate mechanism exists for changing a trait

Consequences of Mutations in Germ Cells and Somatic Cells

• Germ line cell mutations found in ovaries/testes can be passed on to offspring
• Somatic cell mutations occur in body cells but cannot be passed on
• If a mutation occurs in sperm or egg, all cells in offspring will carry the mutation or somatic cells, depending on timing of mutation

Mutation as a cause of genetic variation

• Gene mutations are the original source of all genetic variation
• Though most mutations are either harmful or neutral, they are essential for evolution by natural selection
• Without mutation species would decline and lack variation
• Genes are regions of DNA coding for a protein, and alleles are variations of a gene
• As there are pairs of chromosomes, there are pairs of genes coding for every protein except where chromosomes X and Y are not homologous

Gene knockout

• Gene knockout investigates the function of a gene by changing/deleting it to make it inoperative
• Gene knockout typically happens by doing deletions and insertions in a targeted way
• Gene knockout is mainly used in animal models like mice
• The process involves inserting engineered embryonic stem cells into a blastocyst
• After birth mouse genetics allows the identification of knock-out of full chromosomes
• "Gene libraries" are developed of different strains with different knock outs for research

Limitations of the model

• KO can have many unexpected effects
• Gene knockouts in animals may not = humans
• 15% of gene knockouts are lethal
• There are concerns about gene knockout:
  • genes in animals may not have the equivalent in humans
  • knockouts for same genes can be different in different animals (ex P53)
  • 15% of gene knockouts can be lethal

CRISPR uses

• Bacteria use crisper and an enzyme Cas9 to identify & destroy viruses
• CRISPR-Cas9 is gene editing based on a natural system that exists in many species of prokaryotes, which use it as a defense against viral attacks
• In nature, the main two elements for this system are CRISPR regions within the bacterial genome and the enzyme Cas9
• Clustered regularly interspaced repeats are in a part of the bacterial genome
• Repeats are interspersed with base sequences (2-120 bp) at a crispr locus, call spacers made from Viral DNA - allowing recognition of virus if reinfection occurs
• Repeats - Same base sequence repeated several times in the genome

CRISPR and the genome

• Viral DNA one repeat - one spacer
• Bacterial spacer sequences transcribed into the guide RNA that binds to Cas9 protein used to search for target DNA
• If viral DNA is present endonucleases in Cas 9 complex cuts the DNA
• In order to fix non homologous end joining (NHEJ- more errors) used or homology directed repair ( HDR)
• donor DNA template used for more precice modification (from cell)
• Use of Cas 9 leads to targeted insertions or deletions that creates small indels

Gene editing as a medical device using CRISPR

• Sickle cell anemia has single point mutation where CRISPR used to edit gene
• Edited hematopoietic Stem Cells returned to patient
• Prime editing uses guide RNA that identifies where to edit
• Enymes are inserted into the faulty cell, faulty DNA removeds and replaced

Conserved or Highly Conserved sequences

• Conserved sequences are identical or similar in nucleic acids (DNA or RNA) across species or a group of species
• Highly conserved sequences are identical or similar over long periods of evolution
• Most conserved sequences are in protein coding areas of the genome, which are sequences with a known function
• There are two hypothesis for highly conserved DNA sequences either they have slow mutation or have a vital gene regulatory effect in the cell
• Most conserved sequences are in protein coding areas of the genome Rate of mutation is slow in highly conserved sequences, least in the transcribed strand [template or anhserse ] perhaps better proofreading
• Conserved Sequences in non coding sections of DNA are involved in gene regulahon
• Ribosomal RNA + tENA ave requved by all cells for basic metabolism is consereved
• The protein cytochrome c (cyt c) and its corresponding is highly conserved

Explore the differences between base substitution and frameshift mutations, and the significance of gene mutations at the species level. Learn about gene knockout techniques and their applications in disrupting gene function. Understand the potential mechanisms behind varying phenotypes in knockout lines.