BIOL302- Fundamentals of Genetics and Genomics

Central dogma of Biology

DNA to RNA to protein

DNA structure

Antiparallel double helix, AT double bonded, CG tripple bonded- semiconservative replication, with errors which can be propegated

Start codon

AUG = Met

Stop codons

UAA UAG UGA

Prokaryotic gene structure

Cis regulatory sequence, promoter region where transcription factors bind, termination sequences

Eukaryotic gene structure

Intron & exon, exon is the coding sequence that gives rise to a protein- introns spliced during transcription

Cis-regulatory elements

are specific DNA sequences located on the same molecule as the gene they regulate, whereas trans-regulatory elements are diffusible molecules (like proteins or RNAs) that can travel through the cell to regulate genes anywhere in the genome

Chromosome compaction

• DNA around histones, nucleosomes- further compaction of proteins, tightly looped, and coiled into x shape

  • Affects availability of DNA for TF access

Euchromatin

loosely wrapped regions are accessible to tfs

Heterochromatin

tightly compacted, inaccessible

Loci of regulation

Allele

alternative forms of a gene- wild type v

Genotype

genetic constitution of an organism

Phenotype

An organism’s observable traits

Pleiotropy

a genetic phenomenon where a single gene or genetic variant influences two or more seemingly unrelated phenotypic traits

Polygenic trait

a characteristic controlled by two or more genes working together

Impacts on phenotype

Genotype and External environment, and internal environment of the cell

Phenylketonuria

Example of Phenotype is not affected solely by genotype:

• Lack enzyme to convert phenylalanine to tyrosine

• Can treat with low phenylalanine diet

What does genetic variation arise from?

• Mutation

  • New variation of genes = new alleles

• Recombination

  • Different combinations of alleles

What are mutations

• are a change in DNA seq. that can lead to:

  • Altered gene coding/regulatory sequence

  • Altered phenotype

• Mutant is an individual with altered phenotype compared to an often arbitrarily defined form

Somatic mutations

Non heretible

• Happen in body tissue

• Population of cloned mutant cells

Germ line mutations

• Passed to approximately half of next gen

  • Bc mutations Only affects one copy of a gene

Point mutations

change/add/del of one or few nucleotides

Insertional mutations

insertion of large chunks of DNA- transposable elements

Chromosomal mutations

• losing gaining, or swapping large bits of chromosomes

  • Trisomy 21

Synonymous point mutations

• silent mutation

  • Codons specify the same amino acid

    • Eg. AGG → CGG both coding for Arg

Missense point mutations

• altered codon specifies another aa

  • Conservative

    • Chemically similar aa

      • AAA (Lys(basic)) → AGA (Arg(basic))

• Nonconservative: chem. dissimilar aa

• Consequences depend on location

  • A functional domain will have greater weight

Nonsense point mutation

• aa specifying codon replaced by a stop codon

  • Truncated proteins-

  • Mutation location matters- if early in the sequence, likely strong loss of fxn

Frameshift mutation

• Addition or deletion alters downstream codons by putting them out of frame

  • Chance of having premature stop codons leading to truncation

Regulatory sequence

• Promoter

• Polyadenylation

• Splice site

• DNA replication

Base substitutions

swapping of a base pair

Transition

Purine to purine; pyrimidine for pyrimidine

Transversion

purine to pyrimidine, pyrimidine to purine

What if a mutation falls outside the coding region

• If it falls within an important cis sequence, the mutation can affect transcription of a gene

• Wrong place, wrong time

• A mutation that affects a splice junction can retain introns or splice out part of an exon

Spontaneous mutations

Spontaneous- ‘background’ level of mutations, no purposeful or accidental exposure to mutagen

Mispairing

• Tautomeric shifts allow base mispairing

  • Bc of altered physical form, mispairing happens

Depurination

• lose a purine group-

  • Repair enzymes will slot something in -

    • If not detected and repaired- bp substitutions can occur before replication

Deamination

• loss of nitrogen group

  • C → U is easy to spot and repair

  • DNA methylation can cause problems

    • Locations of 5 methylcytosine are mutational hotspots

Fixation of spontaneous mutation

• Fixation of mutation is when second replication round occurs without repair- the mutation is fixed in the genetic code

• Replication slippage- usually associated with repeated DNA sequences

  • Can lead to additions and deletions

Base analogs

• Can be integrated into DNA in place of nucleotides, less stable forms than bases; shift base-pairing affinities

  • eg. 5 bromouracil (5BU)

    • Causes A/T to G/C or vice versa

Base alteration

• (eg. alkylating agents)

  • Eg ethylmethane sulfonate, adds ethyl group

    • G/C to A/T

Intercalating agents

• mimic base pairs / integrate into double helix

  • Eg provlavin, ethidium bromide

In our environment

• Radiation - eg uv radiation and x rays

• Chemicals - cigarette smoke, barbecuing, benzoyl peroxide

• Infectious agents- HPV, helicobacter pylori

Base damage- uv

• UV light can cause interactions between adjacent pyrimidines

  • Creates this different linking , typically between adjacent thymines

• Creates bulky bend in DNA that can cause issues in replication

Base damage - other mutagens

aflatoxin B1, leads to apurinic site via depurination event

Ames test

• Whether or not a compound is mutagenic/ poses mutagenic risk

• Start with a initial mutation in bacteria that’s hist- , place it on a medium that contains histadine- it shouldn’t grow

• Then you place another potential mutagenic compound, and look at how many reversion compounds you get

• REVERSION is the central concept.

True reversionf

Get a reversion of function- revertant codes for the same aa

Intragenic reversion

restores the reading frame

How to test mutagenic potential in animals

• is add enzymes- see if the compound is mutagenic AND if the metabolic compounds may be mutagenic as they’re broken down

  • Rat liver enzymes (S9) use to see if chemicals that are n

How do organisms deal with mutations?

• Very efficient DNA repair systems

  • Proofreading subunit of DNA polymerase repairs many

  • Reverse mutations

  • Remove altered nucleotides

  • Detect and remove mismatches

  • Paste together broken DNA molecules

Direct reversal (not in humans)

• Photorepair of pyrimidine dimers

  • Performed by photolyases which detects bulky distortions in the helix

Base excision repair

• Removal triggers the AP repair system

  • Ap endonuclease cuts at the AP site

Nucleotide excision repair

• DNA damage where no glycosylase to detect that type of damage

  • Pyrimidine dimers from uv damage

    • Cuts made either side to remove a stretch of 30 nt around damage

  • DNA polymerase uses the template strand to repair the strand

Mismatch repair

In short window of time post replication, when parent/daughter strands don’t line up-

What happens if dna replication stalls?

• SOS repair/translesion repair- bulky additions and/or pyrimidine dimers can lead to stalling of DNA polymerase

  • To let replication to continue, error - prone ‘repair’ polymerase is activated that puts random nucleotides opposite the lesion (leads to mutation itself

  • Goes until regular polymerase can rejoin.

Nonhomologous end joining

• Ends trimmed/ligated back together, which causes deletions

• Error prone

• Helpful when no template to guide correct repair is available

Homologous double strand break repair