Nilson2_mutations Flashcards
Can mutations in all cells be inherited?
No, only mutation in the germline
What is a positive effect of mutations?
They generate diversity → adaptation/evolution
What are 3 general classes of mutations?
Chromosomal mutations → gain or loss of a part of a chromosome
Insertional mutations → insertion of large regions of DNA (ex: transposable elements)
Point mutations → changes in single nucleotides, indels (1 or more nt)
What are different types of base substitutions?
Transition:
- Purine → purine
- Pyrimidine → pyrimidine
Transversion:
- Pyrimidine → purine
- Purine → pyrimidine
Which nitrogen bases are pyrimidines and which are purines?
Pyrimidines → C, T
Purines → A, G
What are consequences/types of point mutations in the open reading frame?
- Synounymous mutations (Silent mutations) → doesn’t change the amino acid it codes for
- Nonsynonymous mutations (Missense mutations) → change the sequences of amino acids
- Conservative mutations → chemically similar AA (K → R)
- Non-conservative mutations → chemically different AA (K → T) - Nonsense mutations → change the sequence of codons to add stop codon
- effect depends on the distance from the 3’ end of the ORF
- Can trigger nonsense-mediated decay → degrades mRNA - Frameshift mutations
- Caused by indels
- Change the translation reading frame for all codons downstream from mutation
What are the stop codons?
UAA
UAG
UGA
What are different effect point mutations can have on a gene’s function?
- WT function (no effect)
- Loss-of-function (partial or complete)
- Hypomorphic → protein retains some funciton or is produced at a reduced level
- Null → protein is non-functional/not produced - Gain-of-function (gene function increased or novel)
- hypermorphic → protein is hyperactive
- ectopic → more protein is made or made in wrong time/palce
- neomorphic → protein gains new function
Why would only half of the F1 indivudals carry a mutation and not all of them?
Because the mutatio could be found in only 1 of the 2 copies of homologous chromosomes
What is an example of a gain-of-function mutant allele?
WT Glycin at 12th position → Valine (G12V)
This mutation locks Ras in active form → blocks hydrolysis of GTP to GDP
What can non-conding region mutations have an effect on?
- Transcription
- Splicing → at snRNP binding site, a mutation could genrate a new splice donor or acceptor
- Stability → in 3’ UTR (RISC/miRNA)
- Translation → at the promotor or enhancer site
- Function
- etc.
What sequence is associated with a splice donor site and a splice acceptor site?
Splice donor = GT (5’/start of intron)
Splice acceptor = AG (end of intron)
What methods can be used to detect if the mutation affects the mRNA transcript or protein levels?
Northern blot → RNA
Western blot → Protein
Ex:
- Early stop codon → short mRNA
- Mutation leading to nonsense mediated decay → no bands in either
- Misense mutation → single AA substitution → no change in either blots
- Mutation in a regulatory region → no bands in either (no transcription)
- Mutation in promotor region → no band in western, but normal northern
What are possible origins of spontaenous mutations?
- Error in DNA replication:
- Base mispairing
- Strand slippage - Spontaneous chemical changes to the DNA → errors in replication
- Depurination
- Deamination
What is DNA polymerase’s error rate?
10^-8 *3x10^9 base pairs → ~ 300 mutation/mitosis → >99% are fixed by DNA repair mechanisms
What are tautomers?
Isomers that differ in the position of their atoms and in the bonds between the atoms
The forms are in equilibrium
Ex: 2 bonds N-N-H become N=N — H
*They exist in equilibrium
How does spontaneous C-T mutation occur?
When G is in its enol form, it interacts with T instead of C (mismatch)
If it is not repaired, in the next replication, CG → TA
What does spontaneous strand slippage lead to during replication?
Lead to indels in repeated sequences:
If the newly synthesized strand slips (extra base loops out) → insertion (in the next replication cycle, the double helix for which the new strand will be a template will have insertions)
If the template strand slips out (extra base loops out) → deletion (in the next replication cycle, the double helix for which the new strand will be a template will have deletions)
*only in repeated sequences, bc template and insertion can stilll be complementary
How do trinucleotide repeat disorders arise?
Arise from expansion of repeated sequence (nt.)
The greater the number of repeats → the greater the chances of slippage → increase number of repeats (cycle) → when reach threshold, phenotype of the disease appears
What are polyQ diseases?
Disease caused by expanded CAG repeats (CAG = Glutamine) → abnormal folding → protein aggregation → neural degeneration
Normal # repeats ~ 4 - 40
Disease # repeats ~ 40 - 80 (depends, up to 300)
What is Fragile X Syndrome caused by?
It is caused be trinucleotide repeat expansion in non-coding region of the FMR1 gene
FMR1 gene = CpG - CGG repeats - ATG + coding sequence
The number of CGG repeats in 5’ UTR affects phenotype, stability, methylation, transcription
CGG < 45 → stable, no methylation, normal phenotype, transcription
55 < CGG < 200 → unstable/prone to expansion, no methylation, normal phenotype, transcription
CGG > 200 → unstable/prone to expansion, affected phenotype, methylation → no transcription
What is Huntington’s Disease caused by?
It is caused by trinucleotide repeat (CAG) expansion in the CODING region of the HTT gene
Number of CAG repeats:
Normal allele < 26
Mutable normal allele 27 - 35
HD allele with reduced penetrance 36-39
HD allele > 40
*greater n → greated slippage
Passed on to progeny → if an offspring gets the to alleles of the parents that have the higher number of repeats → more chances
What is a deamination spontaneous mutation?
Caused by chemical change leading to the transcription from C → U (C-G → T-A)
Cytosine + H2O → Uracil + NH3
*N=C(-C)-NH2 → HN-C(-C)=O
NH2 is replaced by =O
What is a depurination spontaneous mutation?
It has the effect of blocking DNA replication and transcription → can’t base pair
Pentose + Guanine → (apurinic site) pentose-OH at the site where guanine was attached (1’C) + Guanine detached
*Can lead to sequence errors during replication → 10,000 purines lost/cell in a day (usually repaired)
If not repaired → apurinic site (deletion of G) → in the next replication, on the the dsDNA has a single nt deletion
What mutations can be caused by oxidative damage?
- Thymine → Thymine glycol (addition of OH on 2Cs) → no possible base pairing
- Guanine → 8-Oxoguanine → interacts with Adenine (to C) → Transversion (GC → TA)
What is the major cause of oxidative damage induced mutations?
Caused by byproducts generated in the mitochondria by normal aerobic metabolism of molecular Oxygen → ROS
ex: superoxide radicals (- O2-), hydrogen peroxide (H2O2), hydroxyl raidcals (- OH)
By which 3 different mechanisms can mutagens induce mutations?
- Replace a base in DNA (gets incorporated instead of the actual base)
- Alter a base so that it mispaires with another base (ex: alkylating agents)
- Damage a base so that it can no longer base pair with any base (ex: UV light, ionizing radiation)
How can UV light damage bases?
UVB light induces covalent interactions betwe Thymines → if not repaired, blocks DNA replication
How can ionizing radation induce base damage in DNA?
Causes strand breaks → mutations in homologous rejoining
+ Oxidative damage
How can we know if something is a mutagen?
Ames test:
1. Start with cells that have a mutation that makes them unable to grow without histidine (in a plate)
2. Inubate with a media without histidine, but with different potential mutagens
3. If see cell growth → mutation was reverted → the compounds were mutagens (different degrees)
*If treatment of this his-mutant slamonella with a compound increases the number of revertants, then that compound is a mutagen
How can we use the Ames test to assess for compounds that might become mutagenic only when metabolized?
Add liver enzymes to the mixture
How can we use Ames test to better understand the type of mutation a mutagen induces?
Strain 1 = His-transition mutant
Strain 2 = His-frameshift mutat
What is forward genetic vs reversed genetics?
Forward genetics: Phenotype → Genotype
Reversed genetics: Genotype → Phenotype (start with the sequence)
What is the first thing we can look for as evidence of possible exons in a DNA sequence?
Look for open reading frames → looking for long sequence without stop codon?
Why is it complicated to identify protein coding genes?
- Genes are normally have multiple exons (~10 in human)
- Exons can be big or small
- Introns can be big or small → exons can be close or far from each other
- Alternative splicing and/or transcriptional start sites can generate multiple mRNA isoforms
What is codon bias? What can it be an indicator of?
Codon bias can be an evidence that ORF is part of a real gene
Differetn species have different frequencies of synonymous codon usage → condon preference reflects relative tRNA abundance and can be signature of an ORF that’s part of a real gene
Ex: in human, R → AGA, AGG, CGC mostly
in drosophila R → CGC (48%)
in E. coli → CGC (39%), CGU (29%)
*Peak conservation align with exons
What is the best evidence that ORF is part of a real protein coding gene?
If we find a cDNA maps to its exons or an EST
EST = expressed sequence tag → cDNA than has not benn completely sequenced (only parts of both ends
What method can help predict that some sequences of amino acid are related to specific protein?
BLAST similarities
If 3% of the DNA in the human genome represents exons of genes, why does only 1% encode protein sequences?
*Exons + introns ~ 28%
Not all exons of a protein coding gene are in the actual protein:
- alternative splicing
- some stay in heterochromatin state
- long 5’ UTR before start codon (AUG)
- 3’ UTR after stop codon
- some genes might be missing a promotor
What can comparison of sequences within a genome reveal?
Can reveal families of related genes → PARALOGS (2-100 members)
- Can be functionally redundant of have indepent functions
- Can arise through gene duplication during evolution for example (human keratin-associated protein 1 family)
*Involves sequencing just ethe exome → cost-effective, faster
What are the steps for sequencing of only the exome?
- Shear DNA into segments
- Bind exonic DNA to immobiliseed probed and purify
- Elute and amplify exonic DNA
- Sequence exonic DNA
What information can be taken from comparing genomes within a species?
- # different genes affect dog size
- Some dog breeds have higher incidence of X cancer
- Breeds with short legs have en extra copy of FGF4 gene → involved in limb development
- At least 3 genes control length, texture and curl of a dog’s coat
- NOT codon bias
What is Synteny?
Synteny is the conserved order of genes between the 2 genomes → similarities in genome organization between species (at levels of the genes and of the chromosomal rearrangements)
*Overall genome organization including relative order of genes and non-coding regions is also highly conserved between mice and human genome
99% of mouse genes have homologs in the human genome AND 99% of human genes have homolog in the mouse genome
What is Phylogenetic interference?
Study of orthologs and paralogs:
Orthologs → homologous genes at the same genetic locus in different species, inherited from common ancestor
Paralogs → homologous genes at different loci in the same species, arisen from gene duplication
Why do platypus lay eggs?
*Used phylogenetic interference to understand it
Laying eggs required presence of the vitellogenin gene (“egg yolk”)
Chicken has 3 vitellogenin genes, platypus has one → suggests animal closer to platypus that don’t lay eggs lost that gene (platypus didn’t gain it)
How can uni parental disomy occur?
(F1 has 2 father (instead of 1 father/1 mother) chromosome 5, but all the rest is normal)
Non-disjunction in meiosis followed by “trisomy rescue” in the embryo
*the rescued chromosome has to be from the other gamete (to keep uni parentality)
Nondisjunction in Meiosis I → HetUPD (in meiosis I, a gamete with 2 homologous 2x chromatid sisters, 1 gamete with nothing) → both homologs from one parent represented in the child
Nondisjunciton in Meiosis II → IsoUPD (sister chromatids don’t separate) → only 1 homolog from 1 parent is represented in the child (x2 for disomy)
Are all chromosomes equally likely to be found in Uni parental disomy?
No
Mostly maternal chromosomes are disomic
Non-disjunction → very plastic cellular process
Not all chromosomes have the same probability → ex: maternal Chr 16 is by far the most frequent
