Lecture 2: Mutation & Genetic Interactions

Introduction

Focus of this lecture (Lecture 2 Overview):

  • The phenotypic consequences of mutations.

  • Understanding alleles, including dominant and recessive forms.

  • Differentiating between gain-of-function and loss-of-function mutations.

  • Exploring genetic interactions when two or more mutant alleles are present.

  • Defining epistasis and genetic suppression.

  • Introduction to genetic pathway analysis.

Alleles and Their Phenotypic Consequences

An allele is an alternative form of a single gene. For example, ade6-M210 and ade6-M216 are different alleles of the ade6 gene in fission yeast.

Allele Classification in Diploid Organisms:

  • Dominant Allele: An allele that expresses its phenotypic effect even when heterozygous (i.e., when only one copy is present alongside a different allele). Most wild-type alleles are dominant.

  • Recessive Allele: An allele whose phenotype is masked by the presence of a dominant (often wild-type) allele. An individual must typically have two copies of a recessive allele for its phenotype to be expressed. Most mutant alleles are recessive.

Genetic Nomenclature: While there's no universal standard, and rules vary for each model organism, some common conventions include:

  • Gene names are italicized (e.g., ade6, wtf1).

  • Protein names are in Roman type and often start with a capital letter or are in full capitals (e.g., Ade6, Wtf1).

  • Allele designations are typically superscripts:

    • + often denotes a wild-type allele (e.g., Wtf+).

    • - (or lowercase gene name) can denote a recessive mutant allele.

    • ts indicates a temperature-sensitive allele.

    • D can indicate a dominant mutant allele.

Types of Mutations Based on Effect on Function

Mutations can be broadly categorized by how they affect the function of the gene product.

Gain-of-Function Alleles

These mutations result in a new or enhanced activity of the gene or its protein product. They are often dominant.

  • Hypermorph: A mutation that generates either more of the normal protein than the wild-type allele or a mutant protein that is more efficient in its function.

    • Example: The human FGFR3>(G480R) mutation. The FGFR3 protein is a receptor that normally inhibits bone growth when activated by its ligand (FGF). The FGFR3>(G480R) mutation causes the receptor to be constitutively active (always "on"), even without FGF, leading to constant inhibition of bone growth. This allele is dominant to the wild-type FGFR3 allele and results in Achondroplasia, a common form of dwarfism. Peter Dinklage, an actor with Achondroplasia, a condition often caused by a hypermorphic mutation in the FGFR3 gene.

  • Neomorph ("New Form"): A mutation that generates a mutant protein with a new function or causes a normal protein to be produced at an inappropriate time or place (ectopic expression).

    • Example: The Antp>(Ns) (Antennapedia-Nasobemia) allele in Drosophila melanogaster (fruit flies). The Antp gene normally functions to specify leg development in the thoracic segments. The dominant Antp>(Ns) mutation causes the Antp protein to be expressed in the head region, where antennae normally develop. This results in flies having legs growing on their heads instead of antennae. Left (A): Wild-type fruit fly head with normal antennae. Right (B): Fruit fly with an Antp>(Ns) mutation, showing legs growing where the antennae should be.

  • Antimorph (Dominant-Negative): A mutation that generates a mutant protein that not only fails to function normally but also interferes with the function of the wild-type protein produced from the other allele in a heterozygote.

    • Mechanism Example: The mutant protein might bind irreversibly to the substrate of an enzyme, preventing the wild-type enzyme from accessing it.

    • Example 1: The Axin>(Kinky) allele in mice. Axin protein is involved in establishing the body axis during development. Mice heterozygous for the Axin>(Kinky) allele (Axin>(Kinky)/Axin>(+)) have short, "kinky" tails due to the mutant Axin protein interfering with the normal Axin protein.

    • Example 2: The eyeless>(D) (ey>(D)) allele in fruit flies. This dominant-negative allele results from an insertion in the ey gene, producing a truncated protein that interferes with the function of the normal Eyeless protein, leading to abnormal eye development in heterozygotes (ey>(D)/ey>(+)). (Note: Overexpression of eyeless can also lead to neomorphic effects, such as eyes growing on wings or legs ).

Loss-of-Function Alleles

These mutations cause a partial or complete loss of the normal function of the gene product. They are often recessive.

  • Amorph (Null): A mutation that results in a total loss of protein function. This can occur if the mutation prevents the synthesis of the full-length protein (e.g., premature stop codon) or produces a protein that completely lacks function. Amorphic alleles are generally recessive to the wild-type allele.

    • Example: The white (w) gene in fruit flies determines eye color. Wild-type flies (w<sup>+</sup>/w<sup>+</sup>) have red eyes. Flies homozygous for a null allele (w<sup>-</sup>/w<sup>-</sup>) have white eyes because they cannot produce the pigment transporter. Heterozygous flies (w<sup>+</sup>/w<sup>-</sup>) have red eyes, showing the null allele is recessive. Fruit fly eye (wild-type, e.g., w<sup>+</sup>/w<sup>+</sup> or w<sup>+</sup>/w<sup>-</sup>) vs. White (e.g., w<sup>-</sup>/w<sup>-</sup>).

  • Hypomorph: A mutation that results in a partial loss of protein function. This can be due to reduced gene expression (less protein made) or reduced activity of the protein itself. Hypomorphic alleles are generally recessive to the wild-type allele.

    • Temperature-sensitive (ts) alleles are often hypomorphs. The mutant protein functions adequately at a permissive temperature (e.g., 25°C) but loses significant function at a restrictive temperature (e.g., 36°C). The fission yeast orb6-25 allele is an example, showing a rounded cell phenotype at 36°C but normal growth at 25°C.

  • Incomplete Dominance (related to Loss-of-Function): Occurs when the phenotype of the heterozygote is intermediate between the phenotypes of the two homozygotes. This often happens when the phenotype varies continuously with the quantity of the gene product.

    • Example: Snapdragon flower color. If R<sup>+</sup> is an allele for red pigment and R<sup>0</sup> is a null (amorph) allele, R<sup>+</sup>/R<sup>+</sup> flowers are red, and R<sup>0</sup>/R<sup>0</sup> flowers are white. R<sup>+</sup>/R<sup>0</sup> heterozygotes, producing half the amount of pigment, may be pink.

  • Haploinsufficiency (Unusual Dominance for Loss-of-Function): Occurs in diploid organisms when one copy of a wild-type allele is not sufficient to produce the normal phenotype, when the other allele is a loss-of-function (amorphic) mutant. The loss-of-function allele, therefore, appears dominant because the heterozygote shows a mutant phenotype.

    • Example: The human GLI3 gene encodes a transcription factor important for digit specification. Individuals heterozygous for a null GLI3 allele (GLI3<sup>null</sup>/GLI3<sup>+</sup>) often exhibit polydactyly (extra fingers or toes) because half the normal amount of GLI3 protein is insufficient for proper digit development.

Summary of Phenotypic Effects of Mutations:

Mutation Type

Occurrence

Possible Dominance Relations

Loss-of-function:

Amorphic (null)

Common

Usually recessive; Can be dominant in cases of haploinsufficiency

Hypomorphic (partial loss)

Common

Usually recessive; Can be incompletely dominant if phenotype varies continuously with gene product

Gain-of-function:

Hypermorphic (increased activity)

Rare

Usually dominant; Can be incompletely dominant

Neomorphic (new function/place)

Rare

Usually dominant

Antimorphic (dominant-negative)

Rare

Usually dominant or incompletely dominant

Genetic Interactions of Multiple Mutant Alleles

Genetic interaction occurs when multiple genes contribute to a single phenotype, or when the phenotypic effect of an allele at one gene is influenced by alleles at other loci.

Epistasis

Epistasis is a form of gene interaction where an allele at one gene locus masks the phenotypic effects of alleles at another gene locus.

  • The gene that performs the masking is called the epistatic gene.

  • The gene whose phenotype is masked is called the hypostatic gene.

  • Recessive Epistasis: The recessive genotype (e.g., aa) at one locus masks the expression of alleles at another locus (e.g., B_).

    • Example: Coat color in Labrador Retrievers. Gene B controls melanin production (B=black, dominant; b=brown, recessive). Gene E controls melanin deposition in hairs (E=deposition, dominant; e=no deposition, recessive). A dog with the genotype ee will have a pale yellow to white coat regardless of the alleles at the B locus, because allele e (when homozygous) prevents pigment deposition. Thus, ee is epistatic to the B/b alleles.

  • Dominant Epistasis: The dominant allele (e.g., A_) at one locus masks the expression of alleles at another locus (e.g., B_).

    • Example: Fruit color in some squash. Gene Y determines pigment color (Y=yellow, dominant; y=green, recessive). Gene W inhibits pigment production (W=no pigment, dominant; w=pigment allowed, recessive). If a squash has at least one W allele, it will be white, irrespective of the alleles at the Y locus. Thus, W is epistatic to the Y/y alleles.

  • Reciprocal Recessive Epistasis (Complementary Gene Action): Dominant alleles at both loci are required to produce a specific phenotype. A recessive genotype at either locus masks the effect of the dominant allele at the other locus.

    • Example: Flower color in Sweet Peas. Purple flowers are produced only if dominant alleles of both gene A and gene B are present (e.g., A_B_). If a plant is homozygous recessive for either gene (e.g., aaB_ or A_bb) or both (aabb), the flowers are white. This often occurs when genes encode enzymes in the same biochemical pathway (e.g., colorless compound #1 --Gene A--> colorless compound #2 --Gene B--> purple pigment).

Phenotypic Reversion (Suppression)

The phenotypic effect of an initial mutation can sometimes be reversed or suppressed by a second mutation.

  • True Reversion: The original mutated base pair changes back to the wild-type sequence. This is extremely rare.

  • Intragenic Suppression: A second mutation occurs within the same gene as the original mutation, restoring or partially restoring the wild-type phenotype.

    • Example 1 (Frameshift): If an initial deletion caused a frameshift, a nearby insertion (or vice versa) might restore the correct reading frame for a significant portion of the protein.

    • Example 2 (Missense): If an initial missense mutation altered protein structure and function, a second missense mutation elsewhere in the same protein might cause a compensatory structural change that restores function (e.g., by improving substrate binding).

  • Intergenic Suppression (Extragenic Suppression): A mutation in a second, different gene suppresses the phenotypic effect of the original mutation in the first gene.

    • Nonsense Suppressor tRNA: An original nonsense (stop) mutation in a gene (e.g., creating a UAG codon) causes premature termination of translation. A suppressor mutation can occur in a gene encoding a tRNA (e.g., a tRNA for tyrosine). This tRNA mutation changes its anticodon so that it now recognizes the UAG stop codon and inserts an amino acid (e.g., tyrosine) at that site, allowing translation to continue and produce a full-length protein (though it may have one amino acid change). Such suppressors can have genome-wide effects. (a) A nonsense mutation creates a premature stop codon. (b) A mutated tRNA (nonsense-suppressor tRNA) recognizes the stop codon and inserts an amino acid, allowing full-length protein synthesis.

    • Interaction in the Same Pathway: Mutations in different genes that function in the same biological pathway can modify each other's phenotypes. This is a powerful tool for genetic pathway analysis, even without knowing the DNA sequence of the mutations.

Genetic Pathway Analysis using Intergenic Suppression: Case Studies

Analyzing how mutations in different genes interact can reveal their functional relationships and order in a pathway.

Case Study 1: Fission Yeast Cell Cycle Genes (cdc2, wee1, and cdc25) Studies by Sir Paul Nurse and others used fission yeast mutants to dissect cell cycle control.

  • Single mutant phenotypes (temperature-sensitive, ts):

    • cdc2<sup>ts</sup> (hypomorph/loss-of-function): Elongated cells, arrested in the cell cycle. Cdc2 promotes cell division.

    • wee1<sup>ts</sup> (hypomorph/loss-of-function): "Wee" (small) cells, divide prematurely. Wee1 inhibits cell division.

    • cdc25<sup>ts</sup> (hypomorph/loss-of-function): Elongated cells, arrested in the cell cycle. Cdc25 promotes cell division.

  • Double mutant analysis revealed interactions:

    • wee1<sup>ts</sup> cdc2<sup>ts</sup>: Elongated phenotype (like cdc2<sup>ts</sup> alone). Suggests Cdc2 function is essential for division, and removing its inhibitor (Wee1) doesn't help if Cdc2 itself is defective.

    • cdc2<sup>Dom</sup> (hypermorph/gain-of-function) + wee1<sup>ts</sup>: Results in "mitotic catastrophe" – cells enter mitosis too early and die. This is because Cdc2 activity is too high (hyperactive Cdc2 and loss of its inhibitor Wee1).

    • cdc25<sup>ts</sup> wee1<sup>ts</sup>: Cells have a near-normal size. The loss of an activator (Cdc25) is balanced by the loss of an inhibitor (Wee1), leading to a partial restoration of normal cell cycle timing.

  • Pathway Elucidation: These genetic interactions, combined with later biochemical studies, revealed that Cdc2 is a kinase (CDK) that drives entry into mitosis. Wee1 is a kinase that phosphorylates and inhibits Cdc2. Cdc25 is a phosphatase that dephosphorylates and activates Cdc2. The genetic pathway is: Wee1 --| Cdc2 |-- Cdc25 (Wee1 inhibits Cdc2, Cdc25 activates Cdc2).

Case Study 2: Fission Yeast Cell Polarity Genes (scd1, tea1, and gef1) Genetic interactions also helped elucidate pathways controlling cell shape.

  • scd1Δ (null mutant) results in round cells but with some residual polarized growth. Scd1 promotes polarized growth.

  • A scd1<sup>low</sup> (hypomorphic) mutant combined with tea1Δ shows a complete loss of polarized growth (very round cells). This suggests Scd1 and Tea1 act in partially redundant or parallel pathways to promote polarized growth.

  • Introducing a gef1Δ mutation into the scd1<sup>low</sup> tea1Δ background partially reverses the completely round phenotype, making cells more polarized. This indicates a complex interplay where Gef1 might normally inhibit some aspect of polarity that becomes overly active when both Scd1 and Tea1 are compromised.

  • Conclusion: Scd1 acts as a local GEF (Guanine nucleotide Exchange Factor) for the Rho GTPase Cdc42, Tea1 is involved in delivering polarity factors to cell tips via microtubules, and Gef1 acts as a global GEF for Cdc42, all contributing to the establishment and maintenance of cell polarity.

Take Home Messages

  • Mutations can be classified by their effect on gene function (loss-of-function, gain-of-function) and their dominance relationships.

  • Epistasis describes how alleles of one gene can mask the phenotypic effects of alleles of another gene.

  • Genetic suppression occurs when a second mutation alleviates the phenotypic effects of a first mutation.

  • Analyzing genetic interactions between different mutants is a powerful tool to dissect complex biological pathways, as exemplified by studies of the cell cycle and cell polarity.