Lecture 4: Translation (Cont), Mutation types and effects Learning Objectives

Define mutation in DNA

• A heritable change in the base-pair sequence of DNA

• Drives phenotypic variation within species

• The raw material of natural selection

• Random mutations must also be rare

• Too many would likely result in loss of cellular integrity, leading to loss of viability of the species

List the ways in which mutations can be classified and explain what defines each category

Explain the difference in outcomes when mutations occur in somatic cells vs. germ cells

• Somatic cell mutations: Acquired during life and passed to daughter cells

• Germ cell mutations: Occur in gametes and are passed to all cells of offspring

List the different types of point mutations and explain how they may affect gene expression. Identify the multiple functional effects on proteins

• Point mutation: changes to a single nucleotide found within one single gene

• Three types of point mutations within genes:

1. Base substitutions

2. Base insertion: Addition of one nucleotide

3. Base deletion: Removal of one nucleotide

• Base substitutions replace one nucleotide for another

• Two categories of base substitutions

1. Transitions: One purine is swapped for the other purine, or one pyrimidine is swapped for the other

2. Transversions: A purine replaces a pyrimidine, or a pyrimidine replaces a purine

• Note: these effects presented will occur within the open reading frame (ORF)

• Substitution mutations may or may not have an effect on the protein product:

1. Silent: A base substitution in DNA that changes an mRNA codon to another codon specifying the exact same amino acid (due to degeneracy in the genetic code). No change to the amino acid sequence or protein function.

2. Missense: A base substitution that alters a codon so that it specifies a different amino acid. Changes a single amino acid in the polypeptide chain, which may alter protein folding, active site function, or stability

3. Neutral mutations: A specific type of missense mutation where the substituted amino acid has similar chemical properties (e.g., swapping one hydrophobic amino acid for another, like Leucine to Isoleucine) or occurs in a non-critical region of the protein. The amino acid sequence changes, but the overall structure and physiological function of the protein remain unchanged or unaffected

4. Nonsense: A base substitution that converts an amino acid-coding codon into a premature stop codon (UAA, UAG, or UGA). Causes early termination of translation, producing a truncated (shortened) and usually non-functional protein.

5. Insertions or deletions will affect protein product

6. Frameshift: An insertion or deletion of a number of nucleotides not divisible by three in a coding sequence. Completely shifts the triplet reading frame downstream of the mutation, altering every subsequent amino acid and almost always creating a premature stop codon shortly after.

Define indels and unstable trinucleotide repeats and explain the functional effects on proteins

• Indels: Insertions or deletions of multiple nucleotides

1. If done in multiples of 3 within the ORF, reading frame remains intact

2. More likely to significantly effect protein function

• Unstable trinucleotide repeats: Regions within the genome that contain a sequence of 3 nucleotides repeated multiple times

1. Ex. CGGCGGCGGCGGCGG

2. In some cases, the number of repeats is highly variable-different somatic cells within the same individual can have different numbers of repeats

3. If instability of repeat number occurs during gamete formation, the next generation of offspring will have a different number of repeats than parent

4. Known in ~20 human genes, all of which are associated with neurodegenerative disease. Ex: Huntington Disease, Fragile-X syndrome

5. Disease is correlated with too many repeats in the allele

6. Expansions appear to occur in only one germ line

Define loss-of-function and gain-of-function mutations and identify subtypes. Explain how the phenotype of these mutations is related to the biological amount and/or function of the gene product (mostly proteins)

• When mutations affect the phenotype, typically the amino acid sequence of the protein is altered or the amount of gene product is altered

• Loss of function (LOF) mutations: Mutations that reduce or abolish protein activity in any way

1. Mutations may occur in ORF or in regulatory sequences

2. LOF mutation subtypes: Null (amorphic mutations), Hypomorphic mutations, Incompletely dominant, and Dominant (very unusual)

3. For some proteins, biochemical output of the pathway is proportional to the amount of protein present in the pathway

4. Incompletely dominant alleles will produce a spectrum of phenotypes between the extremes. Ex: Three alleles, rather than two, are present in the population and produce flower color. R0 is a null allele, R50 allele produces only half of the typical amount of protein

5. Some pathways are highly sensitive to changes in the amount of functional protein. In these cases, a heterozygote with an amorphic allele and wildtype allele will NOT have the wildtype phenotype called haploinsufficiency. Ex: Haploinsufficiency of the human gene GLI3. The presence of one LOF allele causes the appearance of a 6th digit

• Gain of function (GOF): Mutations will enhance the protein function or amount, confer a new activity on the protein, or express the protein at the wrong time/place.

1. Typically these variant alleles are dominant to the wildtype.

2. Typically these are lethal in the homozygous state because of pleiotrophy (product is active in multiple pathways)

3. GOF mutation subtypes: Hypermorphic mutations, Neomorphic mutations, and Antimorphic mutations (aka dominant negative)

4. Hypermorphic mutations create a higher amount of the typical protein product or create a mutant protein that is more efficient than the typical version. Ex: A hypermorphic allele of the human FGFR3 gene causes achondroplasia. Gene encodes a receptor that responds to growth factor, which inhibits bone growth when FGF is present. Hypermorphic will cause growth inhibition without FGF present. Receptor is constitutively active

5. Neomorphic mutations will create a new characteristic in the organism. Can produce a new protein with a new function. Can cause ectopic expression of the typical protein (expression occurs at the wrong time/place). Ex. Huntington disease. The new allele with more repeats makes a mutant protein, and wildtype version cannot suppress activity. Ex: Antennapedia mutant in Drosophila. A mutation in regulatory DNA causes the production of legs in place of antenna

6. Antimorphic mutations do not have activity of typical protein product but also prevent the typical protein from functioning. Often called dominant-negative mutations. Abstract example. A functional protein requires four subunits, and all subunits are encoded by the same gene. A mutant allele will produce a subunit that will prevent the entire protein from functioning, even if the other three subunits are the wildtype allele. So little functional complex is produced that a new phenotype will result