Bio2C03M2

What are the 3 types of point mutations?

1. Coding-sequence mutations

2. Regulatory mutations

3. Transition/Transversion mutations

4 Coding Sequence Mutations

1. Synonymous: no amino acid sequence change

2. Missense: changes one amino acid

3. Nonsense: creates stop codon and terminates translation

4. Frameshift: wrong sequence of amino acids

4 Regulatory Mutations

1. Promoter: changes timing or amount of transcription

2. Polyadenylation: alters sequence of mRNA

3. Splice site: improperply retains an intron or excludes an exon

4. DNA Replication Mutation (DRM): increases or decreases the number of short repeats of DNA

Transition and Transversion Mutations

• Transition Mutations = Purine —> Pyramidine or Pyramidine —> Purine

  • Ex: A —> G or T—C

• Transversion Mutations: Pyramidine —> Pyramidine or Purine —> Purine

  • Ex: A —> T or C —> G

Which is more common? Transition or transversion mutation?

Transition mutations are more common because it is more likely to substitute a same ringed base pair than a different one

Does an insertion or deletion result in frameshift mutations?

No. If basepairs are inserted or deleted in pairs of three, the reading frame is unaffected

Forward Mutations

Change from wild type allele to mutant allele

Reverse mutation (reversion)

Change from mutant allele to wild-type allele

3 types of Reverse Mutations (reversions) [TIS]

1. True Reversion

2. Intragenic Reversion

3. Second-site Reversion

True Reversion

The mutation restores the exact wild-type DNA sequence

• The codon can be another sequence that still codes for the same type of amino acid

• Ex: UUA (Leu) —> UUC (Phe) —> CUC (Leu)

Intragenic Reversion

A mutation elsewhere in the same gene restores gene function

Second-site Reversion

A mutation in a different gene compensates for the original mutation, restoring the wild-type phenotype

• AKA: supressor mutation

3 Mechanisms of Point Mutation

1. Mispaired nucleotides during replication

2. Spontaneous nucleotide base change (chemical interactions)

3. Mutagens (chemical or radiation)

Mispaired Nucleotides During Replication (Leads to Point Mutations)

Non-complementery base pairing can occur (an incorporating error)

• Without repair, replication leads to mutation

• G:T pairing or C:A pairing (not normal pairing)

Spontaneous Nucleotide Base Change (Depurination)

Loss of a purine, creating an apurinic site

• If not repaired, DNA polymerase will fill the gap with an adenine (A) during replication

• A common way for a G —> A substitution to occur

Spontaneous Nucleotide Base Change (Deamination)

Loss of an amino group from a nucleotide base

• Methylated cytosine (C) can undergo deamination to become a thymine

  • Leads to a mismatch, which when repaired can cause a C-G pair to become T-A

Chemical Mutagens (Lead to Point Mutations)

1. Nucleotide Base Analogs: a chemical with a similar structure to DNA which is then incorporated into DNA during replication and then induces a point mutation

2. Deaminating Agents: removes amino groups (Can stimulate a C-G pair to become T-A)

3. Alkylating Agents: add methyl or ethyl groups to nucleotide bases, causing a distortion in the DNA helix, leading to mutations

4. Oxidizing Agents: oxidizes nucleotide base, usually resulting in a transversion mutation (change to different structure NBP)

5. Hydroxylating agents: add hydroxyl groups to a nucleotide base, usually resulting in a modified cytosine pairing with A

6. Intercalating Agents: molecules that fit between DNA base pairs, distorting the DNA duplex, leading to lesions that may result in frameshift mutations

What is the Ames Test?

A test to verify if a chemical is a mutagen

1. Bacteria is exposed to a chemical in the presence of enzymes extracted from a mammalian liver

   1. Liver enzymes break down toxins into various byproducts for detoxifcation

• Ames test mimics the mammalian liver

• Identifies whether chemical or various detoxifying byproducts are mutagenic

Ames Test Procedure

1. S9 extract (enzyme from liver) is added to 2 different mutant strains of his- S. typhmurium

   1. his-1 = base-substitution mutant

   2. his-2 = framshift mutant

2. S9 bacterial mixture from each strain is spread on one experimental plate and one control plate

3. A paper disk is put on each plate and the test compound is added to the experimental plate disk, but not to the control-plate disks

4. Presence of a significant number of revertant colonies indicates the test compound induces base-substitution mutations

5. The control plates determine the rate of sponatenous his- —> his+ reversion

6. An insignificant number of revertant colonies indicates the test compound does not induce frameshift mutations

Mutations via Radiation

• All high-energy radiation is mutagenic

  • UV

  • X-Rays

  • Gamma Rays

  • Cosmic Rays

• Radiation exposure induces mutations in germ-line, which may get passed on to offspring

  • Mutations occur to the embryonic development gene

UV Radiation (Mutagenic Radiation)

• Thymine dimers are formed from excessive UV radiation exposure

  • Covalent bonds between C5-C6 or C4-C6 of adjacent thymines

• DNA repair mechanisms can repair these dimers

• If not repaired, the dimers can disrupt DNA replication, inducing mutations in the process

*Primary cause for the strong association between excessive UV exposure and skin cancer

Base Excision Repair [BER] (Direct DNA Repair)

Removal of an incorrect or damaged DNA base and repair by synthesis of a new strand segment (nick translation)

• Nick Translation: DNA polymerase initiates the removal and replacement of nucleotides and DNA ligase seals the sugar-phosphate backbone

Procedure:

1. DNA N-glycosylase recognizes a base-pair mismatch and removes the incorrect base-pair, creating an apyrimidinic (AP) site

2. AP endonuclease generates a single stranded on the 5’ side of the AP site

3. DNA polymerase removes and replaces several nucleotides of the nicked strand via nick translation

*Has an over-correction to account for single base-pair mismatch

Nucleotide Excision Repair [NER] (Direct DNA Repair)

Specifically used to repair UV induced damage to DNA (the creation of thymine dimers)

Procedure:

1. UVR AB complex binds opposite a thymine dimer

2. UVR B denatures the DNA around the lesion

3. UVR A departs; UVR C binds and catalyzes 3’ to 5’ cuts

4. DNA helicase (UVR D) helps release the damaged single strand

5. DNA polymerase and DNA ligase fill and seal the single stranded gap

6. Lesion has been removed and the DNA duplex has been restored

*Similar to BER

Mismatch Repair (Direct DNA Repair)

• During DNA replication, the parental strand is usually methylated, while the daughter strand is not

Procedure

1. MutH binds to hemi-methylated DNA

   1. Binds to the methylated parental strand and unmethylated daughter strand

2. MutS binds to a base-pair mismatch and attracts MutL, and the complex contacts MutH

3. MutH cleaves the unmethylated (new) DNA strand, generating a single-stranded gap

4. The gap is then filled by DNA polymerase activity to repair the mismatch

TLDR: deletes the daughter strand and rebuilds it with the correct base-pairing

Transletion Data Synthesis (Direct DNA Repair)

• Last resort method for DNA repair

• Error-prone mechanism

• Unrepaired DNA damage can block DNA polymerase III, causing it to stall

  SOS repair: a repair system in E. coli that is used in response to massive DNA damage that blocks DNA polymerase III

• Activates transletion DNA polymerases (pol V) that bypass these lesions and synthesizes short DNA segments

• pol V has no proof-reading abilities, thus it has a high mutation rate

Double-Strand Break Repair [DSB]

DSBs lack a template for DNA repair

• Can cause chromosome instability, cell death, and cancer

Two mechanisms to repair DSBs:

1. Nonhomologous end joining (NHEJ)

2. Synthesis dependent strand annealing (SDSA)

Nonhomologous End Joining (DSB) [NHEJ]

When double stranded breaks occur, both strands of DNA are trimmed into even “blunt ends” and then rejoined with DNA ligase

• Trimming leads to loss of nucleotides that cannot be replaced

• May lead to frameshift mutations

• Error prone and can lead to mutation

Nonhomologous End Joining Process

1. X-ray or oxidative damage produces double strand break in DNA

2. Ku80-Ku70-PK_cs protein binds to DNA ends

3. Ends are trimmed resulting in a loss of nucleotides

4. DNA ligase ligates the blunt ends to reform an intact duplex

Synthesis-Dependent Strand Annealing (DSB)[SDSA]

After DNA replication, if one chromatid gets damaged on both DNA strands, the intact sister chromatid can help repair

• Error-free process

• Strand invasion offers a template to synthesize new DNA

*Similar to homologous recombination but repairs DNA

Synthesis-Dependent Strand Annealing [SDS] Process

1. One chromatid undergoes a double-stranded break

2. Nucleases digest a portion of the broken strands and Rad51 binds to the undamaged chromatid

3. Strand invasion of the sister chromatid creates a D loop and a replication fork assembles on the D loop

4. New strand synthesis takes place using the available intact strands as templates

5. Partial strand excision occurs; duplexes reform, and strands are ligated

CRISPR Gene Editing and NHEJ & SDSA Procedure

1. Inject an embryo with a plasmid or mRNA to express

2. Guide RNA guids Cas9 (nuclease enzyme) to the genomic target

3. In the absence of a donor template, NHEJ is utilized which can delete the gene of interest (knock-out)

4. In the presence of a donor template, re-insertion of the modified gene occurs (knock-in)

Transposable Genetic Elements [TGEs]

DNA sequences that move within the genome through transposition (facilitated by transposase enzyme)

• TGEs vary in length, sequence composition, and copy number

• All TGEs have terminal inverted repeats on their ends

• Inserted TGE is bracketed by flanking direct repeats (not part of the TGE)

How do TGEs move?

1. Non-replicative transposition: excision of the element from its location and insertion in a new location (cut and pasted)

2. Replicative transposition: duplication of the element and insertion of the copy in a new location (copy and paste)

Transposition Procedure

1. Staggered cuts cleave the DNA strands of the target sequence (cut them into uneven pieces)

2. Single-stranded ends result from staggered cuts of the target sequence

3. The transposable element is then inserted into the target sequence

4. Gaps are filled by DNA polymerase

2 Types of TGEs

1. DNA transposons: transpose as DNA sequences

   • Replicative: copy and past

   • Nonreplicative: cut and paste

2. Retrotransposons: are composed of DNA, but transpose through an RNA intermediate

   • DNA —> RNA —> Reverse transcribed into DNA

   • Reverse transcribed DNA inserted into new location

   • Enzyme reverse transcriptase is used (also used by retroviruses)

Mutagenic Effects of Transposition (TGEs)

TGEs can generate mutations whenever they insert themselves into crucial genetic regions (e.g. coding region, promotor, etc.)

Examples include:

• Humans: hemophilia A, Coffin-Lowry syndrom

• Plants: round vs. wrinkled pea phenotype Mendel studied

• Dorsophila: before 1960 wild flies did not have TGEs, but after 1960, TGEs called P-elements were introduced into flies and they proliferated fast

P-elements and Biotechnology

P-elements: transposable elements (TGEs) found in Drosophila

• Used in a technique to generate transgenic flies (before CRISPR)

Proceduure:

1. Clone gene of interest into plasmid flanked by inverted repeats characteristic of TGEs

2. Inject embryo with plasmid and transposase enzyme

3. Gene of interest will randomly insert itself into the genome of embryo

Epigenetics

The study of traits above inheritance

• Suggests the environment can affect expression of traits

• The acquired environmental influence can then be passed onto offspring

5 features of Epigenetic Modifications

1. Epigenetic modification patters alter chromatin structure

2. They are transmissible during cell division

3. They are reversible

4. They are directly associated with gene transcription

5. They do not alter DNA sequence

Euchromatin

Loosely compacted genomic regions (chromatin)

• Are more transcriptionally active

Heterochromatin

Densely compacted chromatin

• Are less transcriptionally active

Constitutive Heterochromatin

Genomic regions that are always heterochromatin

Facultative Heterochromatin

Genomic regions that switch back and forth between euchromatin and heterochromatin

Position Effects of Epigenetics

1. Transcriptional hotspots

2. Transcriptional coldspots

Heterochromatin vs Euchromatin

• Nucleosomes are made up of DNA wound around 8 histone proteins

  • H2A-H2B dimer + H3-H4 tetramer

• Histone proteins allow DNA to coil around it

  • The coiling of DNA around histones condenses DNA into chromatin

An increases presence of histone proteins means that the chromatin is more compact (heterochromatin) and decreased presence of histone proteins means less compact chromatin (euchromatin)

Acetylation (Chromatin Modifier)

Relaxes histone/DNA interactions by neutralizing positively charged histones

• Histone acetyltransferases (HATs): add acetyl groups to histones, leading to euchromatin

• Histone deactylases (HDACs): remove acetyl groups from histones, leading to heterochromatin

Methylation (Chromatin Modifier)

Can lead to heterochromatin or euchromatin

• Histone methyltransferases (HMTs): add methyl groups to histones

• Histone demethylases (HDMTs): remove methyl groups from histones

Position Effect Variegation (PEV)

Occurs when heterochromatic areas spread into euchromatin, silencing transcription of genes

• Leads to geneotypically wild-type, but phenotypically mosaic (mutant + wild)

E(var) Mutations (Mutation that affects chromatin)

Enhance mutant phenotypes by encouraging spread of heterochromatin

• Mosaic expression leans towards mutant phenotype

• Short for “enhancers of positive effect variegation”

Su(var) Mutations (Mutation that affects chromatin)

Restrict heterochromatin spread, encouraging wild-type phenotype

• Mosaic expression leans towards wild-type phenotype

• Short for “suppressors of position effect variegation”

X-inactivation in Female Placental Mammals

• Occurs early in embryonic development

• Any given cell inactivates either the maternally inherited X chromosome or the paternally inherited X chromosome on a random basis

  • Means all cells in a female’s body are mosaics of two cell types: one expresses the maternal X chromosome, the other expresses the paternal X chromosome

• Also considered an epigenetic phenomenon

Long noncoding RNAs (lnc RNA)

• Long RNA that lack open reading frames

• Plays a role in gene regulation in eukaryotic cells

• Studied in stem cells of mice embryos

• Thought to act as scaffolds that link to regulatory proteins, affecting chromatin structure (modifying it)

What are lnc RNA involved in?

X-inactivation-specific transcript (Xist) is an example of a lnc RNA

• Involved in X-inactivation

What is the process of Xist (lnc RNA) on X-inactivation?

• Xist is present in the X-inactivation center (XIC) on the X chromosome

  • Xist is active in heterochromatic X chromosome

  • Xist is inactive in euchromatic X chromosome

• Xist RNA is produced on the X chromosome that is to be inactivated

  • It spreads along the length of he chromosome and inactivates almost all the genes, silencing the chromosome

1. Xist activation and recruitment of Xist RNA to locations thorughout the chromosome to be inactivated

2. Stable Xist RNA coats the X-chromosome

3. Coat of Xist RNA leads to sliencing and condensation of X chromosome

4. HMTs (histone methyl transferases) are attracted to the RNA coating

   1. H3 and H4 histones are deacetylated and methylated, inactivating the chromosomes

5. Condensed and silenced X chromosome forms a Barr body

Maternal Imprinting

Allele passed on by the mother is inactivated

• Thus, offspring only express the allele from the father

• Only females switch alleles off when passing them on, allowing their children to not be affected

• Only affected males or carrier males can have affected children

Paternal Imprinting

Allele passed on by the father is inactivated

• Thus, the offspring only express the allele from the mother

• Only males switch allele off when passing it on

• Only affected females or carrier females can have affected children

Genetic Imprinting

A change in some genes’ expression in offspring depending on the parent who passed it on

• Can be X-linked or autosomal

• Doesn’t always affect one allele, could affect multiple alleles

• When offspring produce their own gametes, the previous imprinting is erased to ensure their gametes are imprinted in one way

IGF2 and H19 (Imprinting)

• IGF2 and H19 are close to each other on chromosome 11

H19

• Only expressed on maternally inherited chromosome

IGF2 (Insulin growth factor 2)

• Only expressed on paternally inherited chromosome

Mechanism

1. On the maternal chromosome, an enhancer drives expression of H19 and an insulator protein blocks IGF2 expression

   1. Insulator protein basically blocks the enhancer from promoting IGF2

2. On the paternal chromosome, methylation inactivates the ICR and blocks H19 expression

   1. The enhancer drives IGF2 expression

Russell-Silver Syndrome (IGF2 and H19)

Results when both chromosomes display maternal expression patterns

• Insulator protein blocks IGF2

• H19 expression is enhanced

Infants with this condition tend to be born underweight

Beckwith-Wiedemann Syndrome

Results when both chromosomes display the paternal expression patterns

• Methylations causes the ICR to be inactivated and H19 expression is blocked, while IGF2 expression is enhanced

Condition involves overgrowth of tissue

Evidence of Epigenetics in Mice

• A modified agouti gene leads to yellow coat colour and extreme obesity

• Female mice fed a methyl factor rich diet during gestation lead to wild-type offspring, despite inheriting the modified agouti gene

• The methyl factor rich diet lead to increased methylation and silencing of the modified agouti gene

Evidence of Epigenetics in Humans

Long term studies of Dutch citizens that survive a famine suggest that they had increased risk of heart disease, diabetes, and obesity compared to people that did not live through a famine

• IGF2 gene is less methylated in citizens born during the famine (meaning it is expressed more or silenced less)

• Siblings in the same families born after the famine have higher methylation of IGF2

Discontinuous Variation

Traits that are sharply defined and easy to categories (either/or)

• Transmission of these traits can be predictable (ex: 9:3:3:1 dihybrid ratio)

Ex: seed colour and shape in Mendel’s pea plants

Human Height = Continuous Variation

• Phenotypic variation exists on a large numerical scale

• Human height is also an example of a polygenic and multifactorial trait

  • Multifactorial trait = genetic and non-genetic variation affect trait

• Parents transmit a genetic potential that may or may not be met, depending on various influences

Polygenic Traits

Traits that are determined by multiple genes

• Different genes contribute differently to the phenotype

Polygenic Traits in Human Eye Colour

• Human eye colour is determined by up to 15 genes

• OCA2 and HERC2 are two genes with strong influence (major genes)

• Other genes have minor effects on eye colour and are called modifier genes

Additive Gene Effects

• Multiple genes contribute an incremental amount of phenotypic influence

• Alleles of each additive gene can be assigned a value of contribution

• Phenotype is the sum of all allele influences

Goals of Quantitative Genetics

1. How much is the phenotypic variation contributed by genetic factors?

2. How many genes influence the specific phenotypic trait?

3. How much does each of the genes contribute to the phenotypic variation?

4. How do genes interact with each other to influence phenotypic variation?

5. How do genes interact with environmental factors to influence phenotypic variation?

Multiple Gene Hypothesis

The idea that alleles of multiple genes segregate and assort independently and impart additive effects on phenotype

What is the formula for # of phenotypic categories?

2n+1

• n = number of genes

What is the formula to find the frequency of most extreme phenotypes?

(1/4)ⁿ

Allele Segregation in Quantitative Trait Production

Edward East crossed 2 pure breeding lines (1 short and 1 long) tobacco plant

• F1 hybrids were intermediates

• F2 offspring had high variance

• Through additional generations of selective breeding, short and long plant phenotypes were re-established

Conclusion

• Phenotypic variance seen in each generation is due to environmental factors

Threshold Traits

When individuals are affected by a trait at a certain threshold (extreme) of genetic liability

• Genetic liability: alleles that push the phenotypes towards threshold (affected end of spectrum)

• Number of liability alleles in parents can additively produce affected offspring

*Environmental factors can also contribute to reaching threshold trait

Variance (s²)

A measure of the spread of distribution around the mean

s² = sum (x_i - x)²/df (N-1)

• Sum of square differences between each value and the mean divided by degrees of freedom (number independent variables)

Standard Deviation (s)

s = sqrt(s²) = sqrt(Var)

Phenotypic Variance

V_P = V_G (genetic variance) + V_E (environmental variance)

• Genetic variance: genotypic contribution to phenotype

• Environmental variance: environmental contribution to phenotype

Controlling Genetic and Environmental Variability

• Stronger environmental effects = wider variance of phenotypic values

• More diverse population = wider variance of phenotypic values

What is Genetic Variance Made Up Of?

V_G = V_A + V_D + V_I

V_A = additive variance = added effects of all alleles contributing to trait

V_D = dominance variance = contributions due to heterozygous individuals not having intermediate phenotype between two homozygous states

V_I = interactive variance = epistatic interactions between alleles of different genes

Heritability

Measures proportion of phenotypic variation that is due to genetic variation

• Includes broad sense and narrow sense heritability

• Both measures are expressed as a proportion that ranges from 0 to 1

  • 1 = phenotypic variance is strongly explained by genetic variance

  • 0 = little or no genetic variance contributes to phenotypic variance

Broad Sense Heritability (H²)

H² = V_G / V_P

Narrow Sense Heritability (h²)

The proportion of phenotypic variation due to additive genetic variation

h² = V_A / V_P

• High narrow sense heritability correlate with a greater degree of response to selection

Problems with Heritability

1. It is a measure of the degree to which genetic differences contribute to phenotypic variation of a trait

   1. Does not indicate mechanism by which genes control a trait, nor does it indicate how much of a trait is produced by gene action (no way to know many genes are involved)

2. Heritability for a given trait in a population can change

   1. Heritability in one population cannot be transferred to another

   2. Genetic and environmental factors may differ between populations

3. Heritability for a given trait in a population can change

4. High heritability does not preclude environmental factors

   1. High heritability does not dismiss the effect of environment on phenotypic variance

   2. Does not mean the environment plays no role

Broad Sense Heritability in Human Twin Studies

V_P = V_E + 1/2V_G

Caveats

1. Stronger shared maternal effects in MZ than DZ twins

2. Greater similarity treatment by parents of MZ than DZ

3. Greater similarity of interactions between genes and environment in MZ than DZ

Selection Differential (S)

The difference between the mean of the whole population and the breeding population

P-M (mean)

Response to Selection (R)

Depends on the extent to which the difference between the population mean and the mean of mating individuals can be passed on to progeny

• h² = R/S

Selection is strongest when h² = 1

• Tells you that there won’t be variation for the next generations

R = F1-M (mean)

Quantitative Trait Loci (QTLs)

Genes that contribute to phenotypic variation in quantitative traits

QTL Mapping

Mapping QTLs to chromosome regions/linkage groups

• Chromosomal regions are identified through the co-occurrence of a genetic marker with a particular phenotype

Introgression Lines

• Lines derived from backcross progeny by selectively breeding inbred lines together

  • Most of their genome is similar; differences are at key locations of interest