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

Allele

alternative form(s) of a gene

Genotype

genetic constitution of an organism

Phenotype

observable traits (genotype + environment)

How is dominance/recessiveness determined?

Dominance of one allele over another is determined by the

protein products of the allele and the way protein products

of alleles work to produce the phenotype

Muller’s morphs

a set of terms created by geneticist Hermann J. Muller to classify mutant alleles based on how their function changes compared to the normal (wild-type) gene

• either loss of function or gain of function

Null mutation

LOF Muller-

protein is non-functional or not made

Leaky mutation

LOF muller

protein retains some function or only some protein made

Dominant negative mutation

LOF Muller

• non-functional protein affects

  function of normal protein

Gain of fxn mutation

Protein is hyperactive

• More protein made or made in wrong time/place

• Protein gains new function (neomorph)

17

Hypermorphic mutation

Neomorphic mutation

Protein gains new fxn

Dominance vs recessiveness

Is one copy of the wild type ‘enough’ to do the job?

Haplosufficiency

If one copy of wt allele is enough, mutant allele is recessive

Haploinsufficiency

If one copy ISN’T enough- mutant allele is dominant

Incomplete dominance

Eg. ‘bully’ whippets

Codominance

Blood type codominance

Penetrance

Expressivity

Mitosis rev.

Meiosis in germ cells

Meiotic basis for Mendel’s law of segregation

Mammals Sexual alleles

• XX = female, XY = male (hemizygous)

  • Hemizygous, either have the wild type or otherwise

  • Y determines maleness

Pseudoautosomal regions

mediate XY pairing in meiosis- contain few genes that Act like autosomal gnes

Experimental methodology for determining if Y is responsible for maleness

look at XXY and XO progeny

Drosophila sex alleles

• XX = female, XY = male

• However, sex determined by ratio of X chromosomes to autosomes

  • Female if X:autosome ratio is 1:1

    • XXY(female), XO (male)

Bird sexual alleles

• ZW = female hemizygous, ZZ= male

• Still unclear how maleness v femaleness is determined

  • Could be dosage or W dominance

• How do organisms deal with gene dosage for genes on the x chromosome?

• Drosophila: alter gene expression levels, genes on the single X chromosome are hyperactivated in males

• Mammals: One of the x chromosomes is randomly inactivated in somatic cells

Mammal random x inactivation

• Can see in nucleus as structure (get condensed into Barr body)

• Happens early in development, 2 wk post fertilization

  • Can see patches of expression if heterozygous for a gene carried onto the X chromosome

• Differences in Sex development

  • 
    

• Way to characterize genes and hormones in reproductive development- sex dev is different than most others

3 categories of differences in Sex development

Sex chromosome, XY, XX

• Sex chromosome dsd

• abnormality in number of sex chromosomes

  • Klinefelter syndrome (XXY)

  • Turner syndrome (XO)

• XY DSD

• affects genetic males

  • Atypical teste development

  • insensitivity to male hormones

• XX DSD

• affects genetic females

  • ovarian development issues

  • excessive exposure to male hormones

    • Can lead to ambiguous gneitalia or masculinisation of genitalia

Physical basis of Mendelian law of heredity

Chromosomes (diploid case; 2n).

Aberrant euploidy

change in the number of the complete set

chromosomes (e.g., 3 copies instead of 2 → triploid)

Aneuploidy:

plus or minus specific chromosomes in a set (e.g.,

2n+1 → Trisomy 21, Down’s syndrome)

Autopolyploid

Extra set of chromosomes from same species

Allopolyploid

extra set from a different species

Triploids

Causes of autopolyploidy

Somatic consequences of autopolyploidy

Or- larger size, which is common in plants

Germline consequences of autopolyploidy

Infertility in some cases, eg triploid bananas having no seeds