Exam 1 - Lecture 3 Flashcards
Mutations
What is a mutation?
a structural change in genomic DNA which can be transmitted from a parental cell to its daughter cells
What is the difference between germline and somatic mutation?
Germline mutation: present in the gamete
Somatic mutation: present in the somatic cell
What are the types of mutations?
1a. Point mutations (coding)
1b. Regulatory point mutations
1c. Alternative splicing
2a. Deletions/insertions
3. Retrotransposition-related problems
4. Unstable repeat expansion
Substitution mutation
Substitutions will only affect a single codon
Their effects may not be serious unless they affect an amino acid that is essential for the structure and function of the finished protein molecule (e.g. sickle cell anaemia)
silent mutation
A silent mutation has no effect on the phenotype (many exclusions present)
What codons resist mutations more than others?
VAL (valine): GUU, GUA, GUC, GUG
What is an example of missense mutation?
Sickle cell anemia
What is an example of a nonsense mutation?
Familial Hypercholsterolemia (LDLR)
Why does LDLR not make it into the membrane?
when a Protein is too short, It can not fold properly, and Because of that, LDLRs never make it into the membrane and got degraded in ER
How is sickle cell causes?
The change in amino acid sequence causes hemoglobin molecules to crystallize when oxygen levels in the blood are low. As a result, red blood cells sickle and get stuck
in small blood vessels
What is an example of a frame shift mutation?
cystic fibrosis
How is cystic fibrosis caused?
Protein amino acid sequence is shifted, Plus it is too short due to unexpected stop-codon.
It can not fold properly. Because of that, CFTR channel never make it into the membrane and got degraded in ER
What is the function of CFTR protien?
a Cl- channel
What is nonsense medicate mRNA Decay?
Detection of the presence of a premature stop codon in the mRNA that initiates mRNA degradation
What are the 2 forms of damage control for mutated genes?
- mRNAs with pre-mature stop codons got degraded: no protein synthesized
- If truncated protein synthesized, it often degrades in ER before delivery to membrane
What is the importance of damage control?
If the truncated protein is not going to function anyway, We better save resources on its synthesis and delivery to proper location. Also, it might possess novel, cell-damaging function.
What are the types point mutations?
frameshift nonsense missense substitution silent
What does regulatory mutations affect?
Affects promoters, or other regulatory sequences of the gene
What are purposes of duffy blood group proteins?
an antigen
a receptor for chemokines
a receptor for Plasmodium vivax malaria parasites
What are some examples of regulatory mutations?
lactose intolerance
Duffy gene
What is prodynorphin locus (PDYN)?
Encodes opioid neuropeptide precursor for endogenous ligands for opiate receptor: balances “pleasure” genes
What does PDYN do?
- mediate the anticipation and experience of pain
- influence social attachment and bonding behaviour
- affect learning and memory
What does PDYN influence?
Schizophrenia
cocaine addiction
epilepsy
What is DARC
A Chemokine receptor that allows people to be resistant to infection with malaria
What is an example of splicing site mutation?
phenylketonuria
What are large deletions and insertions?
unequal crossing-over events, and retrotransposition
small deletion and insertions
Account for approximately 22% of all known mutations
Often results from slippage of DNA replication
Can be in-frame, if the number of deleted/inserted nucleotides is divisible by three
What does slippage of DNA result in?
This will cause misalignment producing a loop in the nascent strand and increase the repeat length. Alternatively, the same will take place but on the template strand (right) causing a decrease of repeat length
What are the 2 possible outcomes of in-frame deletions?
- Protein is too short, and It is unable to fold properly, and Because of it, protein gets degraded in ER.
Example: ΔF508 1 aa del in CFTR protein - Protein is too short, but It still folds OK. It functions, but not so great
What is Duchenne Dystrophy?
A Missense mutation of 1 bp that changes the conformation of the protein, and renders it non-functional (death at 12-16 years old)
What is Becker Dystrophy?
a In-frame deletion of 2000 bp, rendering the Middle part of the protein gone. Proteins are functional (mild symptoms, live till 60th)
What are large deletion and insertions?
Range in size from 20 bp to 10 Mb (after that they classify as chromosomal abnormalities, as they become visible in light microscope)
Account for approximately 5-6% of all known mutations
What are the ways that unequal crossing-over can occur
between 2 chromosomes, or on the same chromosome
What can unequal corssing-over cause?
May cause a disruption of entire gene, change gene structure, produce fusion of genes
What are the transposable elements in the human genome?
- SINEs (non-autonomous)
- LINEs (autonomous)
What are the results of retrotransposition?
May directly disrupt genes; May provoke illegitemate recombination by homology
3) Contain regulatory sequences
Promoters, Enhancers, Splicing sites, and/or Transcription factor binding sites
These sites can be brought close to the gene
and change its regulation
What are the problems with retrotransposon elements?
33 retrotransposition events identified result in human disease (hemophilia A and B, ß-thalassemia, muscular dystrophy)
What are the ways that transposons can cause problems?
may disrupt integrity of regulatory sites, or the protein sequence
What are the disease that can come about as a result of unstable repeat expansion?
Either suppresses expression of the gene (Fragile X), or it Either results in the production of abnormal toxic product (Huntington disease)
What does unstable repeat expansion cause?
Unstable repeat expansion causes suppression of FMR1 gene and results in Fragile X syndrome
How does expanded repeats cause disease?
Repeats in protein coding sequences = toxic proteins;
Repeats in RNA coding regions = altered RNA function;
Repeats in non-coding regions = reduced transcription or translation
What are the functional classifications of mutations?
- Loss-of-Function mutations
- Haploinsufficiency
- Dominant negative mutations - Gain-of-function mutations
What can loss-of-function mutations cause?
Loss-of-function mutations often cause recessive traits such as halpoinsufficenicey
What is osteogenesis imperfecta (brittle bone disease)?
People with OI either have less collagen (30% of human weight) than normal or the quality is poorer than normal (4 different subtypes of disease)
What is another name for dominant-loss-of-function mutation?
dominant negative or “antimorphic” mutations
What is an example of a dominant negative mutation?
In the cell where one p53 allele is mutated, ½ of p53 molecules are bad; No functional tetramers exist
What is a gain-of-function mutation?
A mutation that confers new or enhanced activity on a protein.
Less common than loss-of-function mutations
what is an example of a gain-of-function mutation?
huntington’s disease
What is spontaneous mutations?
Mistakes occur during DNA replication just before cell division. This is natural error rate of DNA polymerase.
Mutagens increase mistakes to 10–5 (100 thousand) or 10–3 (a thousand) per replicated gene. Mutations overload natural repair systems of the human genome
What are the agents that cause mutations?
- Chemical (different compounds)
- Alkylating agents
- Aromatic hydrocarbons (e.g. benzo(a)pyrene)
- Intercalating agents (e.g. fluorescent dyes)
- Artificial derivatives of DNA bases - Physical (ionizing radiation, UV-radiation)
- Biological (viruses, transposable elements)
benzopyrene
A polycyclic aromatic hydrocarbon with confirmed carcinogenic activity
A product of combustion processes (e.g. tobacco smoking, french fries) Converted by the body from promutagen to mutagen (addition of epoxide group and two OH-groups).
A metabolic product binds to DNA → forms an adduct
(a compound that results from addition).
Presence of adduct change structure of DNA = cause mutation.
What damage is caused by UV Exposure
UV radiation causes formation of the thymine dimers, which block replication, and leads to cancer.
What can result in de novo mutations in germline cells?
spontaneous abortion or baby with genetic syndromes or just nothing: inherited mutation would simply serve as material for evolution
What can result in de novo mutations in somatic cells?
spontaneous cell death or improperly functioning cell (never going to be detected), or CANCER, or just nothing - not inherited: lost in evolution
Where do mutations usually occurs?
MUTATIONS usually happen in the germ-line cells – precursors of sperms and eggs (it means within the mother/father of embry
What is explained by a High frequency of de novo?
The high-frequency of de novo mutations may explain the high frequency of conditions that cause reduced fecundity (autism, schizophrenia, etc).
What is paternal age effect disorder?
A small group of disorders, including Apert syndrome (caused byFGFR2mutations), achondroplasia (FGFR3), Costello syndrome (HRAS),PTPN11 (Noonan syndrome)
What do PAE disorder provide?
these mutations provide selective advantage for mutated cells through dysregulation of spermatogonial cell behavior by growth factor receptor-RAS signal transduction pathway (same way as pre-malignant close outcompetes normal cells)
What pathway does PAE Disorder use?
PAE Disorders Cluster within the Receptor Tyrosine Kinase (RTK)-RAS Signaling Pathway. Signaling downstream of RAS involves the RAF/MEK/ERK (MAPK-ERK branch) (in red) and the PI3K-AKT-mTOR (in green) pathways. The MEK1/2 inhibitor (in red box) specifically blocks the phosphorylation of ERK1/2.
What is are examples of a point mutations?
apert syndrome, dwarfism (increases with paternal age)
What is an example of intragenic deletions?
Duchenne myotrophy (slight increases with age)
What is an example of non-disjunction event?
Down syndrome, 0.3% liveborn are aneuploid (increases with maternal age)
What percent point mutations and deletions are paternal and maternal?
93% of point mutations –> paternal,
87% of deletions –> maternal
What are other types of mutations?
- small insertions/deletions of 1-5 base pairs
- large chromosomal deletions
- translocations
Where is Human HPRT Gene located?
located on chromosome X
What are the possible outcomes if DNA repair doesn’t take place?
- If a mutation is in the germline, it may be passed to offspring (if gamete is not lost) - worst
- If the mutation is in the somatic cells, it might lead to cancer – not good
- In most cases, mutations in the somatic cells do not produce any effect – e.g. cell may die, but we will never fell it (the best case, and most common scenario)
What are the steps in identifying a causative de novo mutation?
- sequence the genome of patient with idiopathic disorder
- select only coding mutations
- exclude known variants seen in healthy people
- sequence parents and exclude their private variants
- look at affected gene function and muational impact
How effective is De Novo?
For 6/9 patients, they were able to identify a single likely-causative mutation
What is the rate of diploid mutations?
175 mutations per diploid genome per generation
Which mutations are more common?
- Transitions are more common than transversions
2. CpG mutate more often than non-CpG
What are the sources of point mutations?
oxidation, deamination hydrolysis, and methylation
What are point mutations?
single nucleotide change
What is the most common point mutation?
transtions: changing purine for purine, or pyrimidine for pyrimidine
pairing is possible due tautomeric shift or inozaing that allows mispairing.
What is transversion?
changing purine for pyrimidine or pyrimidine for purine
pairing is unfavorable but purine-purine pairs are possible (G-A)
What is a random gain of mutations?
low-level, natural cause mutations
Early stages of natural cancer in elderly; spontaneous mutations in germ plasm
What is a forced gain of mutations?
median level; X-ray, chemical carcinogens
Early stages of cancer and germplasm in exposed people
very high rate of mutations.
in cells that lost one or more major mechanisms of DNA repair)
What is the outcome of very high rate of mutations?
Certain genetic syndromes; late stages of almost any cancer
HOW do you CALCULATE RATE OF MUTATIONS IN HUMAN CELLS?
HPRT Assay
What is the normal function of HPRT?
The normal function of HPRT is metabolic salvage of the purines (hypoxanthine and guanine) into nucleotides,
inosinic acid, and guanylic acid
What results from a complete deficiency of HPRT Activity?
too much purunes = Lesch-Nyhan Syndrome (urate crystals + self-Injuring)
Partial deficiency = nephrolithiasis, gouty arthritis, & some neurological manifestations
What happens if cells acquire mutation to HPRT?
if cell acquire mutation in HPRT, it become resistant
to 6-thioguanine compound
what makes individual mutation frequencies
so different?
- Polymorphisms of genes metabolizing carcinogens
- Polymorphisms of genes responsible for DNA repair
- Alcohol consumption and smoking
- Exposure to environmental carcinogens
- Exposure to radiation
What mutations have no influence on HPRT mutation frequencies?
CYP1A1, GSTM1 and NAT2 polymorphisms have no influence on HPRT mutation frequencies
What is the comet assay?
a standard technique for evaluation of DNA damage/repair in biomonitoring and genotoxicity testing
uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. Most often, a non-invasive sample of blood lymphocytes is used
What is the procedure for a comet assay?
- cells mixed with low melting arcose
- immobilized cells on CometSlide
- Treat cells with lysis solution
- sample treated cells with alkali
- sample stained with intercalating dye and visualized epifluorescence microscopy following alkaline electrophoresis, which reveals DNA Breaks
What is the role of lysis solution?
removed membranes and histones from DNA
What is the purpose of alkali?
unwinds and denatures DNA
How is the extent of DNA Damage measured?
The extent of DNA damage is measured by the size and intensity of the tail produced from a single nucleous following electrophoresis
What is the use of FPG?
the lesion specific enzyme formamidopyrimidine N-glycosylase (FPG) was used to access oxidative DNA base damage (it detects alkylation, 8-OH guanine and other oxidatively damaged purines, instead of Physical DNA breaks.)
What is the mutation in lung cancer?
EGRF; treated with IRESSA
What drug was used in china to treat lung cancer?
oltipraz, an inducer of Phase 2 metabolizing enzymes, significantly increased biomarkers of aflatoxin detoxification
What are the purposes of DNA Checkpoint pathways?
control cell-cycle arrest
activates DNA Repair Pathways
control movement of DA repair proteins to the site of DNA Damage
activate Different transcriptional programs
control Telomere length induced cell death by apoptosis.
What is ataxia?
loss of motor control owing to Purkinje cell loss, masked faces; oculomotor apraxia
What is telangiectasias?
Dilated small blood vessels; Skin,
Onset 4 to 6 years
What are the immune deficiencies of ATM?
Recurrent respiratory infections; T-cell function is reduced
Why does ATM defect leads not only to cancer, but also to organismal defects?
- Stalled replication forks needs to be removed during regular cell divisions
- DSBs in normal VDJ recombination and in meiosis also needs repair
- Cells under high-oxidative stress and long-living cells also needs repair – e.g. Purkinje cells (ataxia)
What diagnostic testings are detrimental to ATM Carriers?
ATM carriers develop breast cancer in response to radiation (due to enhanced radiosensitivity of the cells)
Tuberculosis screening and mammograms are harmful for ATM carriers
Where does ATM act on?
ATM acts at the first stage of cellular response (after damage recognition)
How does ATM act?
ATM acts like any other kinase (via phosphorylation). it is part of the DNA-PK family of kinases
What are the substrates for ATM?
Chk2, which is involved in control of both the G1/S and G2/M cell-cycle checkpoints BRCA1 NBS1 Tp53
How is ATM Activated?
ATM is activated by the presence of DSBs (Rapid, within 1 minute, response).
Why do changes in the third base pair have no effect on phenotype?
Changes in the third base of a codon often have no effect on the phenotype due to the degenerate nature of the genetic code
Where is DNA slippage most prominent?
slippage tends to happen in the repetitive nucleotide tracts, e.g. AAAAAA
What is DNA Slippage?
deletion or insertion
What causes larger insertions or deletions?
Often caused by unequal crossing over between homologous sequences
Sometimes are caused by retrotransposition
How is ATR activated?
ATR is activated primarily by stalled replication forks.
How is PK’s activated?
DNA-PKs can autophosphorylation or be phosphorylated by ATM
What does HPRT gene encode for?
Encodes the enzyme hypoxanthine phosphoribosyltransferase
