Module 4: Genes, Chromosomes, and Variation through Mutation

lectures 13-16

central dogma

DNA → RNA → protein

DNA replication

complimentary base pairs, semi-conservative replication

gene structure in prokaryotes

no introns (only coding DNA), regulated by transcription factors in positive or negative direction

gene structure in eukaryotes

has introns and exons, many regulatory sequences and TFs, cis-regulatory sequences are upstream, downstream, near, far, or in introns. 

DNA compaction

DNA is wrapped around histones

what is euchromatin

loosely wrapped regions of DNA (more accessible)

what is heterochromatin?

tightly packed regions of DNA (less accessible)

what is gel electrophoresis used to find?

different lengths of DNA fragments

which techniques are used for DNA/RNA detection?

1. (RT)-PCR → amplification of genomic DNA or cDNA (RT)

2. Hybridization → labelled anti-mRNA probe on gels (northern blot), tissues (in-situ hybridization), or chromosomes (FISH)

which techniques are used for DNA/RNA sequencing?

single-cell RNA-seq → shows what is transcribed in the cell and detects mutations of individual vs. wt

what techniques are used for protein detection?

1. immunohistochemistry → labelled antibodies, western blots

2. protein fusion → GFP, used for in vivo detection

where does genetic variation between individuals arise from?

mutation (new variants of genes) and recombination (different combinations of alleles)

what is a mutation?

a change in a DNA sequence that can lead to an altered gene coding or regulatory sequence and altered phenotype

what is a mutant?

an individual with an altered phenotype

what are somatic mutations?

mutations within body tissue, non-heritable

what are germ-line mutations?

mutations in cells that make gametes, heritable. Most mutations only occur in one of the two alleles (mutant cells are heterozygous) and only half of the gametes will have the mutation

what is a point mutation?

changes or indels of one or a few nucleotides

what is an insertional mutaion?

an insertion of large chunks of DNA (ex: transposons)

what are chromosomal mutations?

losing, gaining or swapping large bits of or whole chromosomes

what kind of point mutations occur in the coding sequence?

synonymous, missense, nonsense, frameshift

where can point mutations happen in the regulatory sequence?

promoters, polyadenylation sites, splice sites, DNA replication mechanisms, etc.

types of base substitutions

• transition → purine to purine, pyrimidine to pyrimidine (A←→G, C←→T)

• transversion → purine to pyrimidine and vice versa (less common)

what are the two types of base pair substitution mutations?

1. silent mutations (synonymous): same amino acid for different codons

2. Missense mutations: different amino acid for different codons (can be conservative or nonconservative)

3. Nonsense mutations: introduction of stop codon due to different codons

what is a frameshift mutation?

when additions/deletions alter downstream codons by putting them out of frame

spontaneous vs. induced mutations

spontaneous : “background” levels of mutations, no exposure to a mutagen

induced: caused by a mutation-inducing agent

what are the different types of replication errors (spontaneous mutations)?

1. mispaired nucleotides (non Watson-Crick)

2. nucleotide base changes

3. nucleotide repeats (replication slippage)

what is depurtination?

a spontaneous mutation that occurs when there is a loss of A or G, and if not repaired base-pair substitution can occur (since it favourably inserts A)

what is deamination?

C → U or 5-meC → T, can lead to base-pair substitutions (5meC are mutational hotspots) 

examples of induced mutations

base analogs, base alteration, damage

what are base analogs?

less stable forms than bases and can be integrated into DNA and cause changes in base pairing (ex: 5-bromouracil causing AT→GC)

what is base alteration?

ethylmethane sulfonate (EMS) adding ethyl groups to bases, causing GC → AT transition

what is base alteration?

when intercalating agents (ex: proflavine and DNA dye ethidium bromide) mimic base pairs, and can cause single insetions/deletions

what can cause base damage?

UV light, aflatoxin B1(apurinic site)

what is the Ames test?

a test used to determine the mutagenic potential of chemicals, and relies on the reversion of mutations. They want to see if the chemical causes the mutated cells to mutate again. 

what is a true reversion in an Ames test?

when the second mutation restores the function of the gene (same amino acid, potentially different codon)

what is an intragenic reversion in an Ames test?

when an additional mutation in another location restores the reading frame of the sequence

what are the DNA repair systems that the body has?

• proofreading of DNA polymerase

• reverse mutations

• removal of altered nucleotides

• detection/removal of mismatches

• paste together broken DNA molecules

direct reversal in DNA repair mechanisms

photorepair of pyrimidine dimers → performed by photolyases and requires light (not in humans)

base-excision repair

DNA glycosylases recognize mismatch, cleave the wrong base (cut out base and surrounding region, use undamaged strand as template and restore missing base pairs)

nucleotide-excision repair

when there are no glycosylases to detect this damage (ex: creation of dimers from UV light). Cuts remove ~30 nt around damaged site, use undamaged strand as template to repair

mismatch repair

• recognized by scanning after replication and due to time-lag of methylation/modifications, the strand without this is excised and repaired

SOS repair

bulky additions cause stalling of DNA polymerase, and in order to allow replication to continue another polymerase that is very error prone takes over, and repairs are made afterwards.

non-homologous end-joining

double-stranded break, where the ends are trimmed and ligated (causing deletions). This is very error prone and only done in cells that are no longer dividing (no template to guide them)

homologous end-joining

double-stranded break, using sister chromatids as a template for synthesis again. 

what are the types of loss-of-function mutations?

null/amorphic: a protein in non-functinoal/not made

hypomorphic: protein has some function/only some of protein is made

dominant negative: non-functional protein affecting function of a normal protein

what are the types of gain-of-function mutations?

hypermorphic: protein is hyperactive, or more protein in made (or in wrong time/place)

neomorphic: protein gains new function

what is haplosufficiency?

in heterozygotes, when only one copy of the wild-type allele is sufficient to do the job (mutant is recessive)

what is incomplete dominance?

when the heterozygote has an intermediate phenotype, and one copy of the allele is not enough (haploinsufficiency)

what is codominance?

when heterozygotes show both phenotypes in a distinct manner

what is pleiotropy?

when one gene affects more than one trait