Organismal genetics Flashcards
Yeast as model organism
Grown in culture/on agar plates, haploid & diploid stages of life cycle, cheap + easy
- can introduce DNA via plasmid via homologous recombination to create mutant
C. elegans as model organism
Worm w/ 4 day life cycle, fast generation, motile, easy identification of defects.
Add DNA via microinjection, reduce gene function via RNAi.
Drosophila as model organism
10 day life cycle, transposable elements allow addition/deletion.
Amenable to mutagenesis + chromosomal rearrangements.
A. thaliana as model organism
Plant w/ 6 week life cycle. Can be transformed via plasmid in agrobacterium.
Amenable to mutagenesis
Mouse as model organism
Similar gene content + physiology to humans.
Amenable to mutagenesis, transgenics, knockouts/knockins, conditional alterations.
Allows for tissue specificity.
Phenylketonuria (PKU)
Autosomal recessive, error in metabolism.
Phenylalanine not converted to tyrosine due to deficiency in PAH -> cognitive defects
Newborns can be screened + mutation found using chemical assay, dietary adjustments for improved cognitive development
Autosomal dominance in pedigree charts
50% chance transmission, must have affected parent, 2 affected people may have unaffected children, males + females affected equally
Autosomal recessive in pedigree charts
Affected person may not have affected parents, all children of 2 affected individuals are affected, males + females affected equally
Penetrance
Probability disease will appear when disease allele present (%)
Expressivity
Range of symptoms possible for given disease e.g. Marfan syndrome can have mild symptoms so hard to diagnose
Cancer can have age-related onset of expression
Pleiotropy
One gene has many functions, can be in many tissues - LoF affects many systems
e.g. Nail-Patella syndrome (NPS), nail abnormalities, absent patella, glaucoma, kidney disease
- LMX1B mutation -> multi-tissue expression
Maternal effect
Genotype/phenotype mismatch. Phenotype depends on gene expression early in development - mRNA + proteins provided by mother in egg.
In organisms w/ delayed zygotic transcription (not mammals)
Allelic series
Different mutations in same gene cause different phenotypes.
e.g. Fibroblast growth factor receptors
receptor domains have many domains + many isoforms due to differential splicing (extra)
FGR3 autosomal dominant disease mutations - great phenotypic variation
Locus heterogeneity
Disease/phenotype caused by dmutations at different loci
e.g. retinitis pigmentosa
e.g. multiple epiphyseal dysplasia
Mainly autosomal dominant, 25% recessive caused by DTDST mutations
Caused by mutations in: COMP (50%), COL9A1, COL9A2, COL9A3, MATN3, DTDST (25%).
-> protein interaction network forms collagen for joint structure development BUT disrupted so abnormal phenotype
Role of COMP in pseudoachondroplasia
Autosomal dominant disorder (1:20,000), more severe than epiphyseal dysplasia. Mutation -> structurally abnormal COMP.
-> example of allelic series
COMP expressed in chondrocytes + tendons
Linkage map
Based on meiosis recombination. Markers farther apart have more recombination.
Tightly linked markers have less recombination
**recombination hot/cold spots so not always linked to distance
Haplotype analysis
Haplotype is combination of alleles present on same chromosome homologue.
- generated from SNP data, SNP combinations inherited + represent individual haplotype
Large scale analysis of haplotypes provides info on regions of DNA differing between populations.
-> disease characteristics
Exome sequencing
Captures all exons to then identify variations (disease causing). Exons selected from total genome + hybridised.
e.g. Primary Ciliary Dyskinesia
Abnormal cilia, lack of function, mutation in 14 different genes.
-> exome sequencing on 2 affected individuals + their parents, looked for coding variants
1 unknown in HEATR2 (likely cause), changes conserved Leu to Pro
Limitations of exome sequencing
- only samples known coding regions
- only identifies sequence changes not chromosomal structure changes
- many variations in each individual so don’t know which causes phenotype
- false positives/negatives due to PCR amplification genes
Next gen sequencing
Allows sequencing of large number of DNA mols in parallel.
- major modifications so don’t need primers
- rapid generation of large datasets
- alignment to reference genome needed
Polygenic phenotype
Phenotype affected by many different genes, linkage more difficult to discover.
Most common diseases are polygenic. SNP markers used to generate haplotypes + GWAS can identify regions associated w/ disease using population haplotypes.
GWAS (pros & cons)
- generate haplotype data from affected + unaffected groups
- evaluate genomic region for association w/ disease
Microarray SNP chip -> Validation -> Replication -> Finemapping -> Functional studies
Advantages: compare large groups + identify regions contributing to variation in phenotype, useful for common disease w/ polygenic basis
Disadvantages: need many people in each group, associated regions often have no genes or unlikely candidates, need functional experiments to demonstrate (association not causation)
What are Mendelian traits?
Traits inherited by offspring from their parents, characterized by discrete units
Mendel proposed that traits are determined by alleles, which can be dominant or recessive.
How can you distinguish between Mendelian and Quantitative traits?
Mendelian traits follow discrete inheritance patterns, controlled by single gene (2 aleles)
Quantitative traits show continuous variation, measurable and influenced by many genes + environemtnal factors.
What does it indicate if a trait disappears in F1 and reappears in 25% of F2?
The trait is recessive to the dominant trait
This follows Mendel’s laws of inheritance.
What is the genetic ratio of offspring from a two-trait cross in Mendelian inheritance?
9:3:3:1
This ratio represents the expected phenotypic outcomes of a dihybrid cross.
What did Francis Galton contribute to the study of quantitative inheritance?
He studied seed size in pea plants and introduced statistical concepts like regression and correlation
Galton’s work laid the groundwork for the biometric approach in genetics.
What is the conflict between Mendelian and biometric approaches?
Mendelian focuses on discrete traits, while biometricians emphasize continuous variation in traits
This conflict delayed the integration of genetics with natural selection theory.
What is the modern theory regarding phenotypic control?
Some phenotypes are controlled by one gene, while others are influenced by multiple genes and environmental interactions
This complexity reflects the multifactorial nature of many traits.
True or False: All phenotypes are controlled by a single gene.
False
Many phenotypes are determined by multiple genes and their interactions.
Fill in the blank: The ratio of offspring from a two-trait cross is _______.
9:3:3:1
What role did Francis Galton play in the eugenics movement?
He was a founder of the movement that led to racist policies and forced sterilization programs
Galton’s ideas, while influential in statistics, contributed to harmful social policies.
Mendelian vs Biometric approach to the effect of mutants on evolution
Mendelians believe variation in discrete characteristics drives evolution (new mutants have large effects)
Biometricians believe that evolution is natural selection acting on continuously distributed characters
What are quantitative traits?
Phenotypes that vary along a range of values, controlled by interactions between genes and environment.
What is quantitative trait inheritance?
The inheritance pattern of phenotypes that can be measured on a continuous scale.
What is the difference between quantitative trait loci and Mendelian trait inheritance?
Quantitative trait loci are genes that cause quantitative traits, while Mendelian traits follow discrete inheritance patterns.
What are major genes?
Genes that produce a detectable effect on phenotype.
How can genes influence phenotypes?
Genes can be additive or dominant in their effects.
What does it mean for genes to be additive?
Each gene contributes equally to the variation in phenotype.
What does it mean for genes to be dominant?
One gene has a more significant effect on phenotype than other genes.
What is heritability test for?
To determine if variation in a trait is controlled by genetics, assessing if relatives are more similar than non-relatives.
Why is classifying individuals with quantitative traits difficult?
Because the effects of many genes are too small to measure individually.
Fill in the blank: Quantitative traits are controlled by interactions between _______ and environment.
[genes]
What are melanosomes?
Cellular compartments that store melanin
Melanosomes play a crucial role in determining pigmentation in humans.
What types of melanin are identified in human pigmentation?
- Eumelanin - black/brown
- Pheomelanin - red/yellow
These two types of melanin contribute to the variety of human skin and hair colors.
What factors contribute to human pigmentation variation?
- Differences in number of melanosomes
- Type of melanin
- Size and shape of melanosomes
These factors are influenced by genetic variants that affect pigmentation.
What is the evolutionary function of pigmentation variation?
- Protection against UV radiation
- Synthesis of vitamin D
Geographic variation in pigmentation serves these important biological functions.
What is the significance of selective mouse breeding?
It generates strains that are homozygous at all loci and have different genotypes at many loci.
What is QTL identification in mice?
It involves identifying genomic variations between strains that affect phenotype.
QTL - Quantitative trait locus
What is the role of F1 in genetic studies?
F1 is produced by crossing two inbred strains and is heterozygous for all loci.
What is a two-bottle choice test in mice used for?
To study alcohol preference behaviour by offering one bottle with water and another with ethanol.
-> D2 consumes less than 1g/kg/day while B6 consumes 10g/kg/day.
What percentage of individuals are affected by alcoholism?
Approximately 5%.
What accounts for 50-60% of the variability in alcoholism manifestation?
Genetic factors.
What does crossing F1 produce?
F2 lines, each mouse having different combinations of homozygous DNA.
What can be inferred if half of the offspring are grey and half are green?
There has been recombination or crossing over.
What is linkage analysis?
Markers which are polymorphic between strains + have a known chromosomal location used:
SSR used, gel electrophoresis to detect SSLP of PCR product -> animals w/ 2 bands are heterozygous.
For SNPs, can detect by sequencing PCR product that amplifies SNP
SSR - simple sequece repeat (microsatellite)
SSLP - simple sequence length polymorphisms
Gene identification
Can narrow down scope of investigation depending on study.
e.g. for alcoholism in mice - expressed in brain, synaptic function, gene w/ sequence change that causes functional change to protein (Stxb1)
How can you confirm that a gene is causative?
Transgenic rescue - add wt allele to mutant & see phenotype disappear
Can use knock-in to generate same sequence change in wt mice + induce same phenotype
Epigenetics
Study of heritable changes that occur without a modification in DNA sequences of genes
- histone modification
- DNA methylation
- nucleosome remodelling (FACT facilitates removal + reincorporation of H2A-H2B dimers)
- non-coding RNA mediation
extra readin**
DNA methylation
Addition of methyl group to cytosine (C) - blocks transcription due to tightly packed chromatin.
- methylated genes generally not expressed
- occurs in mammals + plants
Form of ‘imprinting’ , can change depending on parental chromosome
5’ position of cytosine residues in a CpG dinucleotide
What causes Prader willie & Angelman syndromes?
Micro-deletions on Chromosome 15.
- PWS , short stature, impaired cog development, respiratory distress, obesity
Paternal allele mutated + maternal imprinted (silenced) -> loss of SNRPN expression - AS, developmental delay, hyperactivity, impaired cog function, seizures
Maternal allele mutated + paternal imprinted (UBE3A gene)
What is the epigenetic reprogramming cycle?
- imprint establishment in gametes
- matured and then maintained in fertilisation
- imprint read into differentiated cells
- erased in primordial germ cells
Differentiated gametes undergo widespread epigenetic reprogramming
Zygotic genome activated at 2 (mice) or 8 (humans) cell stage.
In mammals - 2 cell lineages of trophectoderm + ICM.
ICM -> forms epiblast & primitive endoderm
ICM + TE have different degree of methylation. ICM forms embryo + TE forms placenta.
Describe the process of somatic cell nuclear transfer
Cloning e.g. Dolly the Sheep 1996
Nucleus removed from adult cell, oocyte transferred w/ no nucleus.
Implant it into host mother -> nucleus/DNA supplied from adult w/ imprint in place
- electric current applied so nucleus fuses w/ empty egg
4% results in birth of live young
How does large offspring syndrome arise?
Often unhealthy live young from cloning.
- respiratory distress, 2-fold increase in birth weight
- skeletal, immunological + placental defects
Caused by improper placental development due to imprinting defect.
- abnormal X chrom inactivation in females + imprinting (far less methylation) defects both genders
Is epigenetics the cause of clone abnormalities?
Cloned animals used in natural mating -> offspring do not show defects (LOS)
They have same genotype but not same epigenetic imprinting SO yes.
Gene expression changed -> determines survival & phenotype
Histone modifications
Chromatin organisation formulates gene expression pattern.
Epigenetic signatures vary by cell type & in disease.
Chromatin & miRNA environment dictates cellular gene expression
What was Barkers impact on epigenetic inheritance?
Mapped areas of infant mortality + low birthweight in UK 1910s, analysed death rates due to Cardiovascular + type II diabetes 1970s -> correlation w/ deprivation
Hypothesised nutrient conditions during pregnancy affect health in adult life -> permanent metabolic changes (thrifty phenotype)
Programming - caused persistent physiological + metabolic changes which cannot be compensated by adequate nutrition after birth
How did the Dutch Hunger Winter (1944-45) support Barker’s hypothesis?
Maternal starvation during pregnancy limits intrauterine growth of offspring -> higher CV + diabetes rates
-> placental abnormalities + maternal stress
Offspring had children w/ low birthweights -> not found w/ siblings not born in famine
What are the epigenetic changes linked to early development during a famine?
Individuals exposed to famine in gestation -> reduced methylation of IGF2 gene at age 60.
Vs siblings not conceived in famine had higher levels of IGF2 methylation
SO uterine environment encodes epigenetic state during development
- maternal obesity increases risk type II diabetes in child
monozygotic twins discordant for type II diabetes
What are the effects of diet on epigenetic state in rats?
Fed normal and low protein (LP) diet during gestation + lactation.
- Hnf4a polymorphisms confer type II diabetes risk
- Hnf4a expression reduced in LP diet rats - persists throughout life
- Hnf4a levels normally decrease in old age
LP diet induces DNA methylation + histone acetylation on Hnf4a enhancer region -> changes expression throughout life
Transgenerational inheritance
Refers to altered phenotypes in offspring through beyond F2 generation to F3.
- need to eliminate effect on gametes to confirm
- offspring exist as germ cells in grandmothers pregnancy
- can study w/ animal models
Multigenerational exposure can be seen in grandchildren (F2) but no further.
What is the experimental evidence for transgenerational inheritance?
Folate deficiency - folate is carrier of methyl groups needed for cellular metabolism.
Mttr gene needed for normal metabolism of folate + methionine
Mttr mutation (gt) - insertion leads to folate metabolism deficiency
Analysed gt heterozygote intercrosses:
- phenotypes did not correlate w/ maternal or offspring phenotypes
- 45% zygotes showed abnormal phenotype in development
- abnormal phenotypes present in all offspring genotypes equally (F2)
SO hypothesised maternal + grandparental genotype might correlate w/ offspring
How did they determine if Mttr mutation is an example of transgenerational inheritance?
Analysed pedigree charts - pregnancies w/ abnormal litters always had mutant grandparents
- global DNA methylation levels disrupted in mutant adult livers
- Wt littermates show disrupted methylation levels in adult liver
Wt pre-implantation embryos w/ mutant maternal grandparent transferred to wt female for development
Wt embryos w/o mutant maternal grandparents used as controls
-> found congenital defects only in offspring w/ mutant maternal grandparent
Defects persisted for at least 5 generations -> transgenerational
Pleiotropic genes/proteins
Have mutliple functions.
Tend to generate lethal phenotypes early on
SO mosaics, temp sensitive mutants + conditional systems used
Notch homologues flies vs mammals + functional domains
Flies have 1 notch
Notch 1 + 2 highly conserved (similar to flies)
Notch 3 has fewer EGF repeats + no TAD domain
Notch 4 has lost NLS, no TAD + fewer EGF repeats
Notch is signalling mol -> involved w/ proliferation
Forward genetics approah summary
Phenotype -> genotype
- Use existing mutants/ create random ones
- Cross mutants w/ each other to detrmine is on same gene (complementation test)
- Map where gene is (linkage analysis + positional mapping) + obtain its sequence
Can then find out expression patterns, isoforms, compare to database, infer functionfrom phenotype or dtermine molecular nature of mutants
Working hypoethesis can then be tested: biochemica/cell based assay, find partners or targets, engineer new mutants (KD, KO)
Reverse genetics approach summary
Developed after genome projects (human 2003) + RNAi
(CRISPR-Cas9)
Use gene/protein sequence to:
- search database for info on protein domains/homologs
- find out expression patterns
- look for isoforms
- engineer KD or KO, look at phenotype
Working hypoethesis can then be tested: biochemica/cell based assay, find partners or targets, engineer new mutants (KD, KO)
Finds phenotype encoded by a gene
What are Muller’s morphs?
LoF: Amorph (none), hypomorph (reduced), antimorph (antagonistic to wt)
GoF: Hypermorph (increased), neomorph (novel)
Many mutations often change enhancer elements (EEs) so transcription altered
Characteristics of A- & hypomorphs
- Often recessive (unless on X), as normal allele can compensate for loss
- can show dominance if in haploinsufficient genes
-> only if protein works in protein complex OR quantity of protein is critical for function
Characteristics of antimorphs
Interfere w/ wt function + more severe than 1 amorph
Common in proteins w/ binding partner (receptor/dimer), mutation sequesters partner protein -> non-functional
Characteristics of hypermorphs
- high transcription or failure to degrade transcript/protein
-> constitutively active protein (e.g. receptor always bound regardless of ligand)
Characterictics of neomorphs
Novel function
- commonly due tp chnage in expression pattern (mutation in reg sequences)
- also due to translocations -> produce chimeric fusion protein
Example morphs in Notch
EGF-LR repeats: ligand binding altered -> amorph (cant bind), hypomorph(less efficient), possible neomorph (new ligand)
LNR repeats: cleavage altered -> amorph (no cleavage), hypermorph (cleavage w/o ligand)
one NLS: not directed to nucleus -> hypermorph
ANK repeats: prevents regulatory binding -> hypomorph (no +ve partner in nucleus), hypermorph (cant bind -ve reg in cytoplasm)
PEST: less Notch degradation -> hyermorph
Transposon
For insertional mutagenesis
Mobile DNA elements that can jump ~50% human genome
- use cut & paste mechanism
Has 2 inverted repeats (IR) at start + end (complementary to each other, 15bp)
Also has gene encoding tranposase (inc promoter) -> cut + paste
-> has both restriction endonuclease + integrase function
tranposase ofetn imprecise
semi parasitic/viral origin
Non-autonomous TEs
TEs that have lost genes for transposition.
Use transposase from another related TE.
Useful for engineering - has space to insert a COI
- can have nromal/mutant gene, reporter gene, promoter + marker for integration
- transposase from vector 2 binds IRs on vector 1 -> structure folds + trnaposase cuts host DNA
- COI inserted randomly in progeny genome
COI is construct of interest
Transgene
Exogenous gene added into experimental system that can be expressed under its own OR different promoter.
P-elements for mutagenesis in flies
DNA transposons in flies.
COI w/ weak promoter + marker for integration (w+) can rescue mutant phenotype (w-/w- or w-/Y)
Can insert into germline cells - coinject 2 vectors into w- blastoderm.
All females selected + crossed to w-/Y males
-> orange eyed flies contain TE<w+> , partial rescue in transformed cells
How to map TEs for gene identification
- digest DNA w/ restriction enzyme
- re-ligate to form circular DNA
- digest again w/ P-element specific r. endonuclease
- PCR w/ primers that bind to inverted repeats (ulta specific)
- can then sequence PCR product + determine insertion by comparison w/ genomic database
What are the different transposable elements?
- DNA transposons e.g. P-elements
- RNA intermediate transposons/ retrotransposons
^ autonomous
- Non-autonomous TEs for engineering
retroviruses used in zebrafish + mice, more technical limitations than RNA transposons
+ve used to identifiy cancer genes
-ve used in lab to infect blood + mammary cells (leukemia)
Sleeping beauty
modified SB transposon - tool of choice for insertional mutagenesis in vertebrates for cancer gene discovery
- infects all tissue type
- more controllable (infects subset of cells in whole tissue)
- can create several mutations in one cell, tumuour results from ~6 mutations in cancer genes
Rescue experiments w/ TEs
Reverse genetics approach.
- wt copy introduced as COI in a TE or retrovirus
COI has wt version w/ integration marker behind a ubuquitous promoter (e.g. HSP binds sigma32 in RNA pol to initiate transcription, CAG or PGK)
-> see if phenotype rescued
Phenotypic vs molecular markers for mapping chemical/radiatin mutants
Phenotypic - alleles w/bvious phenotypes
Molecular - geneome maps established using polymorphic molecular landmarks
SSLP: simple sequence length repeat polymorphism (microsatellite)
SNP: short nucleotide polymoprhism
How are recessive mutations mapped w/ molecular markers?
- Mutant hom organism selected (m/m), DNA + SNPs extracted at desired locations.
- Choose mapping stock + cross to mutant
- Backcross F1 to mutant stock
- Select m/m organisms in F2 + sequence SNPs
m linked to nearby SNP, will segregate w/ it in meiosis -> homozygous m/m also homozygous for nearby SNP
How are dominant mutations mapped w/ molceular markers?
- Select M/+ instead of m/m & cross to mapping stock
- In F1, select M/+ not m/m, the backcross to mapping stock
- M/+ selcted in F2, look for 100% progeny
Always backcross to mapping stock
M linked to nearby SNP so will segregate w/ it in meiosis.
Mapping is iterative process
How can TEs be used to detect and study gene expression?
Investigating expression patterns gives insight into gene function.
Enhancer trap screens - started in flies
- TE random insertion allows detection of expression controlled by nearby EEs
- EEs work independently or in combination
- EEs can be located far from gene sequence or in introns
P-element enhancer trap screen in flies
w-/w- embryos injected w/ 2 TE vectors (transposase + COI), weak promoter, integration marker (w+) & reporter gene not present in organism (lacZ, GFP)
female w-/w- crossed to male w-/Y -> transposition cuases orange eyed flie w/ TE <w+>
Can check proximity of insertion to EE by checking expression partter of reporter gene.
e.g. lacZ compared in wt vs m-/m-
-> TE insertion revealed vestigial expression controlled by 2 separate EEs in wing
KO, KD & KI
Knock out - delete gene/promoter or critical portion of it, marker replaces all or great part of endogeneous gene/promoter
Knock down - delete small portion of gene/promote, marker replaces small part gene/promoter
Knock in - add construct to genome or replace a gene, DNA(mutation/reporter gene) often fused to marker
Describe KO generation
Gene of interest deleted.
2 integration markers
- 1 to replace gene or promoter (M1)
- 1 to ensure integration via HR (M2)
Typically M1 and M2 code for proteins that are sensitive to drug treatment: e.g. M1 procures resistance to drug (e.g. antibiotic resistance gene), while M2 makes the cell die when exposed to drug (e.g. HSV-TK makes cells sensitive to Ganciclovir).
NB: M1 can also be a reporter gene, e.g. GFP.
Describe the 2 steps to making KIs
Double replacement method w/ +ve & -ve selection -> introduces subtle mutation w/ residual exogenous sequence
1) replace chosen exon w/ reporter gene (GFP) using KO -select all cells w/ GFP, apply drug -> all cells w/ no M2 will survive
2) Replace reporter gene w. mutant DNA, select cells w/ no GFP, apply drug -> cells w/ no M2 will survive
CRISPR-Cas9 for targeted gene editing
Cas9 - helicase + endonuclease w/ 2 ‘scissors’
Guide RNA - matching sequence ~20nt, includes PAM sequence (NGG for Cas9) which is very common, secondary loop structure -> only specific segment binds.
NHEJ -> INDELS resulting in premature stop codon (KO)
HDR (homology directed recombination) requires shorter homologous sequences than HR -> can do 2 cuts for larger insert (KI)
HR less effective - relies on mitosis for effective function, needs more flanking DNA
Short vs long range HDR
Short - Cpf1 (uses sticky ends), most efficient but only for short fragments (GFP) 10bp-2kb, requires short single strand donor DNA
Long - Cas9 (uses blunt ends), same as HR mechanism but more efficient as cut stimulates repair to occur, less homology sequences needed to work
insert up 6kb (0.5-15kb homology needed)
How can CRISPR be used to make transgenic mice?
similar to IVF
Edit single cell embryo + implant morula.
Does not need ES cell culture or chimera - Cas9 infects single cell embryos, guide RNA + donor RNA/DNA -> KO/KD/KI
- one generation process (quicker)
85-95% success, $250 min, only 4 weeks, can be done in house, several mutations possible per mouse
vs HR, 50-55% success, min$25,000, 6 months for 1 mutation
How can a p53 mediated DNA damage reponse be induced?
CRISPR cuts DNA.
Elevated p53 can induce apoptosis, surviving cells can mutate p53 in response to selection pressure & turn into cancer (dangerous in vivo)
Off target Crispr effect can be toxic to cells, any edits are permanent
p53 is guardian of genome (oncogene when mutated)
Function of CRISPR-deadCas9
Catalytically inactive but can bind specifically + irreversibly to DNA.
Can inhibit transcription if sitting on promoter - KO effect but no gene deletion.
dCas9 can be fused to another protein which it delivers via guideRNAs- can regulate transcription by gene regulation (activator/repressor domains) OR epigenetic modification (DNA methylase/demethylase)
repressor domains like KRAB -> zinc finger-like domain that induces chromatin compaction (HDAC recuritment)
Types of screening for genome-wide CRISPR
for Cas9:
CRISPR KO screening - looks at phenotypic effect of tretament where gene is KO (amorph)
dCas9 & fusion:
CRISPRi - interefence screening (KD -hypo), looks at phenotypic effect on essential genes
CRISPRa - activation screening (hyper), effect when gene overexpressed
requires library of sgRNA - can be transduced into cells whihc are then ‘treated’
Principle of conditional systems
Induce recombination or expression in a given space + time
2 transgenic constructs introduced using TEs, HR, CRISPR-Cas9 depedning on organism:
1) driver line - has tissue/cell specifier (drives TF/recombinase expression)
2) responder line - has inducible system (responds to recombinase/TF)
- require crossing 2 established transgenic lines. Transgenic constructs are in all cells of the animal, but only produce desired effect when animals crossed.
can add extra time specifier
Cre-loxP conditional system
Tool from P1 bacteriophage, used in yeast + mice -> deletion mutations (+ inversion & translocation)
- Cre recombinase catalyses recombination between 2 loxP sites
- Orientation of loxP sites dictates combination result
-> circular DNA
Cre is under control of specific promoter (driver)
- where Cre is expressed, construct is floxed/deleted
useful for pleiotropic or lethal genes
Temporal control of Cre-loxP
Cre fused to modified oestrogen receptor that only reacts when tamoxifen present (WHEN), not to endogenous estradiol.
Tamoxifen binds receptor + moves it to nucleus w/ fused Cre.
Floxing happens in cells:
- where Cre is expressed
- when Tamoxifen provided
UAS-GAL4 conditional system for inducible transcription
GAL4 binds UAS sequences in yeast - activates transcription.
No GAL4 or UAS in flies - so introduced into 2 separate trasngenic line using P-elements that contain :
<w+, GAL4> & <w+, UAS +COI>
Not targeted insertion so copy of transgene added
Activation & control of GLA4 drivers
TE insertions just behind EEs are selected - ubiquitous in all cells.
GAL4 can be behind an HSP (if P<w+, hsp-GAL4> inserted randomly) -so promoter only activated when temeprature elevated to 29C temporarily
Can combine GAL4 + GAL80(TS) driver:
GAL4 inserted behind specific enhancer (WHERE)
GAL80 inserted behind ubiquitous promoter (e.g. Tubulin)
change in temp determines WHEN (see footnote)
At 18C, GAL80TS is expressed in all cells and where GAL4 is expressed, it binds to GAL4 and prevents it to activate transcription at UAS site.
When temperature rises at 29C (WHEN), GAL80TS is inactivated (change of conformation due to temperature) and stops biding to GAL4, letting GAL4 activate transcription at UAS site.
Spatial-temporal induction w/ Tet
Similar to UAS-GAL4, tTA expression driven by specific promoter & binding to TRE (WHERE)
tTa inactivated when drug added (tetracycline or doxycycline), ON -> OFF
vs rtTA which only binds TRE if Dox added (OFF ->ON)
TRE - tet response element
tTA - tet controlled transactivator
Phenocopy
Effects comparable to mutant phenotype but notdue to mutations
Caused by RNAi (KDs & KOs)
What effect does RNAi have on RNAi expression?
RNAi blocks/inhibits expression of chosen gene.
Delivered via synthetic mol or inducible plasmid (short lived action) through virus thatcan insert genome.
e.g.
- worms given dsRNA in bateria they feed on
- plants, dsRNA applied to cell culture or via virus (TMV)
- flies, dsRNA injceted in developing embryo + made inducible by UAS-GAL4
- Mice, viruses used to introduce dsRNA to embryonic stem cells, inducible via Cre-loxP or Tet systems
- Useful in organisms not amenable to other genetic approaches (worms)
- Effect seen on whole organism in worms + plants
Pros & cons of RNAi
Pros:
- KD all normal copies of a gene -> homozygous effect (good for polyploid plants)
- Worms demonstrate RNAi effect easily in all cells
Cons:
- effects variable for different organisms/genes, can only show effect in subset of cells
- can show cross reactivity + an off target effect (KD other RNAs w/ similar sequences)
- does not work in yeast, xenopus or zebrafish
What is the Pax6 gene?
Mutations cause murine small eye + human anirida phenotypes.
- it has a paired box domain
- expressed in brain, spinal chord + all stages of eye development
eyeless homolog in flies discovered, a.acids highly conserved (especially DNA binding domains)
- ey2 and eyR (2 existing spontaneous mutations)
Part of pax gene family
Cre-loxP used to study Pax6 function in mice
Cause + effect of ey2 mutation
In situ hybridisation experiments w/ eyeless RNA probe , stage 15 embryos stained for eyeless
- ey2 is hypomorph
Sequenced ey2/ey2 flies -> spontaneous transposon insertion in intron between exon 2 & 3
- intron has an EE so insertion prevents regulatory binding
Residual transcription must be due to another EE
Same for eyR - but has different TE
How can ey2 be rescued?
- Te insertion <wt eyeles & promoter>, randomly integrate into genome
- <UAS-wt> crossed to <eyeless - GAL4 driver line>
*need 2 UAS-ey constructs for full rescue of ey2/ey2
</UAS-wt>
Can also be misexpressed in wrong location -> ectopic eyes on wing, antenna, leg + body.
- eyeless master gene that controls eye development
Mice + Human Pax6 can subsittute for eyeless in rescue/misexpression experiments.
How can GAL4-UAs be used to study eyeless transcription?
In flies:
[eyeless-GAL4 driver] stock mutated using EMS screen -> obtain mutations eyeless regulatory regions
Cahnges intranscription monitored by crossing flies to UAS-lacZ/GFP lines, specific point mutations identified
In mice:
- used HR KI to replace parts of promoter region + fused to lacZ
- tested which modification altered lacZ expression
EMS - ethyl methanesulfonate
Why is drosophila used as a model for development?
- rapid life cycle (10 days) -> large numbers & easy/inexpensive to maintain stock
- easy to obtain mutants -> chemical, x-rays, TEs in males, infrequent genetic redundancy
- well studied organism -> detailed physical map from polytene chromosomes, sequenced genome, linkage analysis given detailed genetic maps
Key features of drosphila genome
Has XY determination + 3 autosomal chromsomes.
- chrom4 very small w/ few genes + no meiotic recombination
- no meiotic recombination in males
Dominance/recessive depends on encoded phenotype
e.g. Curly is dominant by Cy/Cy is recessive lethal in embryos
Deficiency & duplication dosage effects
Df - loss of region of chromosome that removes copy of gene of interest
Dp - reintroduction of wt gene, Dp(vg+) rescues -/- back to wt phenotype
(defines mutant as LoF)
Used to identify LoF mutations
Hypomorphic & amoprhic fly mutants
Hypo - wa/wa leads to orange eyes (partial LoF), weaker phenotype (more red) than wa/Df
- wa has more function than Df
A - w1/w1 or w1/df leads to white eyes (complee LoF)
Haploinsufficieny in flies
Notch
N/+: N is amoprhic allele - dominant function as 1 copy of wt insufficient to maintain normal function
N/Dp - N phenotype completely rescued
Antimorphic fly mutations
Negative activity - antagonise wt gene copies
e.g. ebony (e) - dominant negatives by competing w/ (+) reducing wt gene product activity
Can interrupt tetrameric complexes OR prevent domain interactions w/ susbtrates
Hypermorphic fly mutations
GoF e.g. Elipse in EGF receptor -> increased signalling
Disrupts eye formation.
How can interactions between mutants be measured?
Complementation test using recessive, LoF mutants -> can see if 2 mutants on same gene or not.
Complementation groups worked out using phenotype observed.
Analysis cn then be used to mpa mutations
- use deficiencies in chromosome compared with lethal mutants on another to see if outcome is viable or not
- need to know endpoints of DFs
e.g. salivary gland polyten chromosomes in Drosophila
Heteroallelic combination
when two different mutant alleles are present at the same locus in a diploid organism
e.g. Hypermorhic Elipse complemented by hypomorphic allele despite being mutations on same gene
-> intragenic complementation
if assumptions not satisfied
Dumpy gene in flies
Comeplex gene locus
Has dpo/dpo oblique & dpv/dpv vortex pehnotypes.
Intagenic complementation can arise when different parts of protein have different functions.
OR different mutations lie in dfferent control regions needed for expression of gene in different tissues
makes 1MDa protein
Mutagens
Alkylating agents - ethylmethanesulphonate (EMS) -> very efficient, induces point mutation
Radiation - X-rays + gamma-rays -> chromosomal rearrangements, less efficient, easier to map
P-elements - transposons -> insertions, non-random, low frequency, easy to map
Syncitium
Single cell has many nuclei - very different mechanism to vertebrates
Acquired identitiy along AP axis (3 thoracic segments)
Become primordial germ cells (polar) - forms cellular blastoderm
What did Nusslein-Volhard + Wiechause study?
Development via genes invovled in defining AP axis
Looked at stereotypical sgemented structures - bristly + naked cuticles
AP axis defined at single cell resolution
Principles of recessive screen for recessive lethal mutants
Need to use F2 genetic screen.
Unknown heterozygote mutant males produced in F1, then crossed to wt females.
Then self cross F2 progeny -> identify those carrying embryo lethal mutations (a-/a-)
BUT cannot distinguish +/+ from a-/+ so a- easily lost from stock.
SO use “balancer chromosome” e.g. CyO
-> homozygous cn flies mutated + F1 males cross to females w/ balancer chromosome
-> orange eyes selected + wt rejected
-> F2 progeny self crossed w/ cn phenotype form each line + identify embryo lethal mutants
If mutated chromosome carries recessive lethal mutation then all viable progeny will have curly wings
no recombination in male meiosis
Saturation genetic screen
Aims to generate many mutant lines so there is high probability of collecting relevant mutations
- can colect dead embryos + screen for phenotype of interest (AP defects)
Complementation testing then used to differentiate mutants to comp groups
Why is a balancer chromosome useful?
Allows distincton between a-/+ & +/+ so a- not lost from stock
- carries many inversions which suppress recombination
- carries dominant mutation w/ visible phenotypic marker (Cy)
- carries recessive lethal mutation
- also can have recessive mutation w/ visible phenotype (cn, orange)
CyO is a 2nd chromosome balancer (carries Cy, cn mutations)
Want to end up with a-/Cyo, Cy, cn in our screen -> stable stock (viable w/ Cy phenotype)
a-/a- is lethal
CyO/CyO is lethal
Maternal effect mutations
Bicoid (TF) mutants end up w/ 2 abdomens instead of head, thorax + abdomen
So early development of embryo in drosophila depends on maternal genotype
Cytoplasmic transplant experiments:
- drosophila germ lines have 1 oocyte + 15 nurse cells connected via sytoplasmic bridges
- nurse cells secrete bicoid mRNA into oocyte
- diffusion or bicoid mRNA restricted by binding cytoskeleton -> diffusion gradient
SO nuclei can ‘read’ position in embryo + transcribe genes accordingly after nuclei division
Not good model for vertebrates where cell dividion occurs from 1st nuclear division
Gap gene mutants in drosophila
Zygotically expressed
Several segments of embryo missing.
Gap genes encode TFs regulating gene expression
e.g. Giant & Kruppel gap genes regulated by bicoid gradient -> needed for AP axis formation
Pair rule mutants in drosophila
Zygotically expressed
Missing alternate segments - expressed in narrow stripes in affected segment + encode TFs
e.g. Even skipped & Fushi terrazu
Even skipped has complex regulation by +ve & -ve acting DNA binding proteins (Hunchback +, Giant -)
Segment polarity mutants in drosophila
Zygotically expressed
Missing parts of segment - expressed in narrow stripes down to single cell width, regulated by pair rule genes.
Frequently encode signalling pathway components (more homology w/ vertebrates)
e.g. Wingless & Engrailed
Human homologues mutations (upregulation Wingless + Hedgehog) associated w/ cancer
Defective Hedgehog in humans -> cyclopia
Homeotic mutations in drosophila
Zygotically expressed
Alter identity of segment not number - selector genes so initiates gene expression that confers segment identity.
Family of genes (evolved by gene duplication) that encode TFs + have homeobox domain (60 a.acid helix-turn-helix DNA binding domain)
- fits into major groove of DNA
Regulated by gap + pair rule genes -> determines combinations of homeotic genes expressed
e.g. Ultrabithorax (Ubx)- haltere becomes wing (LoF - identity changed to more anterior segment)
What does the Ed Lewis model suggest?
Homeotic genes repress function of next most anteriorly expressed gene.
Ubx LoF - region of expression reduced, so does not repress Antp (extra wing produced)
Ubx GoF - region of expression progresses more anteriorly, Antp function turned off (haultere duplication + no wings)
Similar effects seen in co-linear order of Hox gene expression in vertebrates.
e.g. Human HoxD13 mutation has extra toe
Drosophila eye as a model system
Model for signalling pathway regulating photoreceptor differentiation.
~800 ommatidia (highly ordered), each one comprised of 20 cell types w/ specific roles + locations
- identity of each cell easily recognised from position (flies lacking R7 unable to moved towards UV light - forwards behavioural screen)
Both sevenless (sev) and Bride of sevenless (Boss) are LoF causing loss of R7
-> form ligand receptor pair which controls R7 fate
Boss & sev have no other developmental function
Enhancer screen function
Type of modifier screen.
identify genes or regulatory elements that increase or enhance the expression of a particular phenotype
Synergistic interaction - if 2 mutations in same pathway then phenotype will be greater than expected from simple addition of phenotype
Suppressor screen function
Type of modifer screen
Mutation in 1 gene pathways suppresses phenotypic consequences of a mutation in another pathway component
What is Ras?
Highly conserved GTPase protein - molecular switch, biochemically activated byt RTK in mammals.
- GTP/GDP exchange factor
Son of sevenless (sos) is homologue of yeast cdc25 which activaes Ras
What is drk?
Downstream of receptor kinase
- homologue of mammalian grb2
Is adapator protein between RTK & sos -> allowing recuritment & activation of Ras.
Binds Pro rich sequence in sos, binds phosphorylated Tyr on sev.
What is GAP and how was it identified?
GTPase activating protein - stimulates enzyme to convert Ras to inactive formation (dephosphorylates)
Suppressor screen for negative regulators of signal used - temperature screen perofmed but with not enough sev to produce wt phenotype
How is Ras linked to cancer?
Human Ras is an oncogene - mutation to Val locks it in GTP bound conformation
Similar mutant (RasV12) engineered into Dros R7 precursor cells -> viable but extra R7 cells
RasN12 locks Ras in GDP bound confromation -> dominant negative by competing w/ wt Ras to bind upstream regulatory proteins
-> loss of R7 cells
Epistasis as a basis for modifier screens
Used to determine if gene function in same pathway + order of function
- uses 2 mutations w/ opposite phenotypes
e.g. hypermorphic GoF mutation of receptor (b) -> always activated SO increased response
- does not matter if ligand (a) mutated or not
b mutation is epistatic to mutation of a as presence of ligand is not relevant to activity of mutant receptor encoded by b
Phenotype of b* = a* + b*
How are mutations that suppress contitutively active sev screened for?
What is Raf?
iMutant sev is GoF, extra R7 cells.
Suppressor screen using EMS mutagen identified Raf as downstream effector of actuvated sev receptor.
Raf is a ser-thr kinase homologous to mamallian MAPKKK
- activated by RTK signalling
- has oncogenc version (always active)
- extra R7 when mutant expressed in Dros eye
How are downstream components of Raf screened for?
Constitutively active form of Raf expressed.
- mutations that suppress phenotype must be downstream
Dsor - drosophila homologue of mammalian MAPKK
rolled - drosophila homologue of mammalian MAPK
Dsor biochemically shown to phosphorylate/activate rolled.
Mosaicism by transposon mobilisation
Maize mutations caused by TE insertion - altered pigment phenotypes. Insertions of Ds in pigment gene -> colourless kernals
- Ac presence allowd Ds to jump out of gene, so back to Wt
OR retrotransposon (LINEs & SINEs) make up large proportion human genome - could be transposition competent
- linked to human disease: neurofibramatosis, MSD, haemophilia
Mosaicism by loss of heterozygosity
+/- –> -/-
can occur by partial/complete chromosome loss OR mitotic recombination (sister chromatid exchange)
Mitotic recombination
Abnormal, caused by radiation -> chrom breaks + then recombined
- sister chromtaid exchange
Two daughter cells can either have same parental genotype OR different non-parental genotypes (hom mutant + hom wt)
Why is mitotic recombination really useful in drosophila?
Can study consequences of recessive mutations that would otherwise be lethal at early dev stage.
Spontaneous MR rate too low - can be increased FLP/FRT system (geenrates MR at defined spot in chrom, high efficiency):
Transgenic stocks created carrying FRT sequence + FLP recombinase
- heat shock induced FLP expression allows induction of clones at defined dev stage
How can the clone generated by MR be located?
Use visible recessive markers on chromosomes - phenotypic marker must be appropriate for tissue + be distal to FRT site
e.g. white- in eyes, loss of GFP (Deltex - ZFD protein - endocytoses cell surface Notch)
Notch and Delta as examples of cell autonomy in expression
N & Dl bind each other + regulate differentiation
N-/N- mutant clone differentiates extra neural cells - shows autonomy -> phenotype of N-/N- not rescued at clone boundary by present of wt N in adjacent cells.
Dl-/Dl- mutant clone differentiates extra neural cells - shows non-autonomy -> extra neural cells only arise in centre of clone, those at boundary rescued by wt Dl+ in durrounding cells
Cell autonomous: gene product required in cells where it is expressed e.g. signalling receptor, TFs
Cell non-autonomous: gene product not needed in cells where it is expressed e.g. ligands for signalling receptors.
Use of MR in genetic screens
Inducing heat shock FLP can induce some pathces of cells homozygous for mutant - can then screen for phenotypes.
- can screen for recessive phen in adult F1 screen
- can create precise deletions, useful for complementation analysis
- can detect late mutation effects otherwise lethal at earlier stage
Can also help identify tumour suppressor genes - loss of heterozygosity induced by MR caused tumours.
e.g. mammalian homologues, lats mutant in mice -> soft tissue + ovarian tumours
Contrast to labour intensive F2 screens by Vollhard + Wieschaus
Use of MR in lineage analysis
Lineage analysis - negative marking
MR can generate negatively marked clones
e.g. GSC marked by absence of lacZ staining
In mammals: engineered Tamoxifen inducible recombinase used
e.g. recombination removes STOP transcription sequence, switching lacZ gene on
Notch signalling and lateral inhibition mechanism for undertsanding neural development
Lat inhibition signal refines neural potential.
- set of precursor cells acquire neural or epidermal cell potentials
- neural cells inhibit other cells around them becoming neural cells.
Neurogenic phenotype if lat inhibition absent - caused by Notch or Delta mutants
dominant gene mutation which is recessive for other phenotypes.
Genetic screens to identify factors involved in axonal guidance
Describe robo + comm
Saturating large scale F2 screen to detect disruptive mutations in axon guidance pathways.
Axons can either go along AP axis or across midline.
2 opposite phenotypic classes - roundabout (robo) & commisureless (comm).
Robo is cell mem protein that detects repulsive signal to prevent midline crossing (axon guidnce in CNS)
Comm is a protein required to keep robo levels low in cells that cross midline, axons can overcome repulsive signal + cross over
Increased robo - axons move away from midline
LoF robo - axons move closer to midline
Genes involved in synapse formation
Dros are good MNJ model - large size, easily accessible + ammenible to different studies.
Temperature sensitive shibire mutant (dros dynamin) reveals dynamin required for synaptic transmission (vesicle recycling)
29C not recycled -> paralysis
18C dynamin able to recycle vesicles
How is drosophila used to uncover mechanisms in memory by Benzer?
Benzer - pioneer of genetic behaviour research
- electrically shocked flies at tube A + used learning index to see if they remembered to go to other tube B.
- screened for mutant lines w/ learning defect, identified dunce mutant.
Dunce encodes drosophila cAMP phosphodiesterase
How is drosophila used to uncover mechanisms in memory by Tully and Yin?
Tully set up massed trial + genetic analysis identified phases of memory
- used CXM drug (protein synthesis inhibitor) to detrmine that LTM requires protein synthesis
Disruption of cAMP pathway similarly affected STM.
Yin:
Used temperature inhibition of TF CREB to show that it blocked LTM but not STM
- use of temp sensitive Notch mutations -> it is required for LTM formation as is regulates Klingon (cell adhesion protein expression
How is drosophila used to study sleep?
Behavioural assay screen set up.
Dopamine uptake mutant found to reduce sleep.
- fumin is spontaneously occurring mutant -> highly active flies at all times of day
- fumin gene cloned + identified as uptake transporter of dopamine
- dopamine signalling implicated in drosophila sleep regulation
fumin mutants show no rebound period after deprivation + instantly rejoin circadian rhythm
sleepless and shaker mutans on drosophila sleep
Chem mutagenesis + TE insertion screens used.
shaker - 1/3 amount of sleep + reduced lifespan, encodes VG ion channel
sleepless - reduced shaker expression, encodes cell surface protein similar to snake neurotoxins
*indicates 2 component regulatory mechanism
Gal4/UAS system for sleep studies
Conditional expression of Na+ channel.
Each Gal4 expressing line crossed to a line which express Na+ channel in response to Gal4.
- identify which Gal4 lines increased sleep + identify regions of overlapping expression
Also used same Gal4 driver to express temp dependent ion channel - flies fell asleep at high temps 31C
Euploidy
Organisms that have multiples of normal chromosome set.
e.g. diploid, triploid, tetraploid
Monoploidy
(n), an organism belonging to a normally diploid species which has only one set of chroms.
Zygotes fail to develop due to deleterious receessive mutations.
If hey survuve to adulthood, normally sterile (no normal meiosis)
Autopolyploidy
Type of polyploidy
Intraspecific - multiple chromosome sets due to hybridisation within a species.
Can arise naturally by doubling 2n complement to 4n (meiotic error), or be artifically inudced in a lab
Odd numbered polyploids mostly infertile - inbalanced gametes.
Tetraploids (4n) can be fertiles as balanced gamates are generated
^meiotic pariring
Allopolyploidy
Type of polyploidy
Interspecific - multiple chromosome sets due to hybridisation between different species
- must be very closely related
e.g Karpachenko’s radish + cabbage
- 2 different species so gametes not recognised, needs polyplooidisation event in Brassica for (18+18)
New species as cannot reproduce w/ eiter to give fertile offspring
- allopolyploidy major force of plant evol
banana only sterile polyploid plant (3n), 33 chromosomes
Polyploidy in animals
In nature: more common in invertebrates but amphibians, reptiles + fish exhibit it, also seen in hepatocytes + 1 rodent (viscacha rat)
In lab:
- drosophila triploids + tetraploids
- yeast can have various levels of polyploidy
Haploidy and diploidy
Haploid (n) - fungi + human gametes
Diploid - human cells (somatic) or yeast cells after cell fusion + mitosis
Aneuploidy
Changes in parts of chromosome sets.
Do not generate viable offspring, but can be tolerated well in some species.
In diploids:
2n + 1 (trisomic)
2n - 1 (monosomic)
2n - 2 (nullisomic)
In haploids:
n + 1 (disomic)
Caused by non-disjunction/mis-segregation in anaphase I or II
- anaphase I mistakes more profound
- humans + animals, it is usually fatal
Monosomy
2n - 1
Missing one copy, usually deletrious.
All autosomal monosomy die in uterus (humans), if XO instead of XX, tolerated but causes Turner’s syndrome
Only trisomic + monosomic autosomal aneuploids in chrom 4 of drosophila survive.
Trisomy
2n + 1
Extra copy of chromosome, deleterious or causes abnormal phenotype.
In humans most ddie in uterus:
- Patau (chr 13)
- Edwards (chr 17)
- Downs (chr 21), least deleterious
XYY & XXX are fertile, XXY (Klinefelter) is tolerated due to X chromosome inactivation
Gene balance & gene dosage effect
Aneuploidy generally more dleterious than aberrant euploidy.
- Euploidy ratios all 1:1
- monosomy 2:1, trisomy 2:3
mRNA level directly proportional to gene copy number - transcription related to amount of protein
SO normal physiology depends on proper ratio of gene product
IF reduction in gene copy number causes abnormal phenotype, then it is haploinsufficient
Why is gene balance necessary and how can it be altered?
Majority of proteins function in complexes.
Aneuploidy causes unlabanced stoichiometry in protein complexes.
Accumulation of subunits can be toxic to the cell
e.g. high levels of undimerized B-tubulin is lethal, even low levels disrupt MT assembly
Aberrant euploidy conserves the gene/protein balance
In waht 4 ways can chromosome structure be altered?
Deletion
Duplication
Inversion - important source of cariation in human lineage (human chr2 from chimp 2A + 2B)
Translocation
Can be caused by either:
- DNA breakage + repair
- crossing over between repetitive DNA
Describe reciprocal translocation
Parts of chromosome arms swapped between non-hom chromosomes.
Meiosis segregation in heterozygte translocation carriers -> chromosomes try to bend to try and pair w/ homologous counterparts -> quadrivalents
Alternate segregation (50%)-> 2 viable T type gametes + 2 viable wt gametes, balanced dyads
Adjacent segregation (50%) -> all dead gametes due to unblanaced dyads
-> segregation leading to viable or dead gametes is random
Human reciprocal translocations diagnosed genetically by semi sterility
Describe Robertsonian translocation
Non-reciprocal where 2 acrocentric chromosomes fuse near centromere region w/ loss of short arms.
-> forms large metacenrtic chromosome + fragment from small arms that fails to segregtae & is lost
Causes Down syndrome: extra info of chrom 21 is fused to chrom 14 .
Skewed meioctic pairing can cause Down syndrome, monosomy in autosomal chrom can cause lethal gamete
Role of translocations in cancer
Can relocate a proto-oncogene to a new promoter region.
e.g. Burkitt lymphoma - chr 14 promoter always active translocated next to MYC TF activating proliferation genes on chr 8
-> MYC always expressed
Can form a hybrid gene
e.g. chronic myelogenous leukemia (CML) - hybrid oncogene from translocation of chr 9 + 22
-> permanent protein kinase activity
Cause uncontrolled cell proliferation
Paricentric inversion
Changes gene order on chromosome
e.g. CDEF becomes CEDF
During segregation, dicentric bridge breaks as chromosomes try to resolve loop structure
-> 1 normal product, 2 deletion products, 1 inversion products
Pericentric inversions
Can move centromere comparative to a gene.
e.g. AB-CD becomes ADC-B
Chrosomes trying to anneal w/ partners causes inversion loop
-> 1 normal product, 2 duplication/deletion products, 1 inversion product
e.g. A or D duplicated after meiosis I
Larger inversion w/ crossing over inside inversion loop -> problems w/ gametes
Describe the 2 tyes of deletions
Inragenic - small deletions within a gene, can inactivate gene (similar to null point)
Multigenic - several genes missing, severe consequences
-> genetic imbalances
Can exhibit pseudominance.
pseudominance - recessive allele shows dominance due to deletion of dominant alleles on corresponding homologous chromosome
How can deletion be identified/mapped?
IN meiosis: failure of pairing between normal segment + its corresponding homolog deleted segment -> deletion loop
Can be viewed in polytene chromosomes in drosophila
Effect of deletions in humans
Dri du chat syndrome: het deletion of tip of chr 5
- microencephaly, metal retadation
- low fatality
William syndrome: 1.5Mb deletion of chr7 homolog (17 genes)
- learning disability, CV disease
- frindly + social personality, good language skills
*thought to be caused by unequal crossing over between repetitive sequences -> haploinsufficiency
Describe the different types of duplications
Tandem - duplicated regions adjacent to each other
Insertional - extra copies somehwere else on genome
Segmented - large scale, most of the duplication is disperesed but some may be in tandem
-> important substarte for nona-alllelic recombination
duplication loops can be formed in meiosis - homolog duplicated segment cannot pair
How can duplications affect Drosophila phenotype?
Alter gene dosage e.g. Drosophila Bar eyes
How can duplications affect humans?
We are 99.9% same but vary in our copy number variation (CNV)
e.g. CNV of AMY1 - people w/ high starch diet have more copies of salivary amylase (AMY1) than those w/ low starch diet.
-> positive selection on a copy number variable gene
+ showed functional relationship between AMY1 copy number & amount of amylase protein in saliva
Whole genome duplication event (changes chromosome number) is usually followed by extensive genome reshaping - changes to chromosomes structure + number.
Identification of conserved genes as a method of comparative genomics
Identify homologs:
orthologs - genes inherited from common ancestor (same locus in differenet species)
paralogs - genes arose from duplication event (different genetic loci in same organism)
Identification of conserved synteny regions as a method of comparative genomics
Chromosomal rearrangements can cause loss of synteny so conserved synteny can indicate functional relationships between genes
Macrosynteny - mouse + human genome have large syntenic blocks in common (human chr 17 w/ mouse chr 11)
Microsynteny - DAL clusters in yeast, DAL used to convert allantoin/allantoate to ammonia in low N conditions
- DAL genes clustered on same chrom region in Saccharomyces + Candida (colocalised)
All genes must expressed simulatensouly for degadation of allantoin.
synteny regions - blocks of co-localised genes on chromosomes of different species
Whole genome duplications as a method of comparative genomics
Can look at known & proposed polyploidy events in eukaryotes.
Humans speculated to have undergone 2 WGDs.
Saccharomyces had WGD event - proven.
Found that duplicated regions were created simultaneously by single tetraploidisation event.
- 50 of duplicated regions maintained same oreinatations w/ respect to ceontromere
- no triplicate clusters
- none of clusteres are overlapping
c. 100mYa WGD event is root of yeast evol.
New S. cerevisiae (NCYC505) is crypto tetraploid (16 chromosomes) + pre-WGD species K. waltii has 8 chromosomes
How is WGD an important source of evolutionary material?
Responsible for genetic redundancy
e.g. yeast genome ~6000 ORFs but <20% genes essential
Genetic redundancy: implies that 2 or more genes carry same biological function + that a mutation in 1 of these genes has little or no effect on overall phenotype, as other would function as backup copy.
So redundant gene is dispensable + can freely mutate:
- sub-functionalisation
- neo-functionalisation
- deleted/lost
Even in absence of WGD, 50% of genes in a genome would be expected to duplicate once every 35-350 million years
C. elegans have widespread gene duplication - twice number local duplications as Drosophila
1/3 largest clusters duplicated gene are olfactory recpetors (7TM GPCRs)- 1/3 of which are peudogenes (non-fucntional)
-> aenahnced survvial rate as increased genetic pool may be beneficial dependning on selection pressure
Problems with genetic redundancy
Redundant genes should be lost eventually due to accumulation of dleeterious mutations (not evolutionarily stable).
So need to break symmetry between duplicated genes by sub or neo-functionalisation.
Examples of neo-functionalisation
New function
fbf-1 & fbf-2 are gene pair in germ line development of C. elegans - encode closely related RNA binding proteins that reciprocally modulate one another to regulate size of mitotic region
citrate synthase in yeast: CIT1 & CIT3 in mt are active in TCA cycle. CIT2 is peroxismal form - active in glyoxylate cycle
- protein functions in novel cellular context (localisation function)
Examples of sub-functionalisation
Sub-functions complementary to other copy
lin-12 & glp-1 receptors in C. elegans have overlapping + seprate functions.
if only lin-12 mutated, larvae doe not die (post-embrionic defects)
- glp-1 has retained lin-12 functions
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Haploinsufficiency
One copy of gene is not sufficicnet alone to maintain original fitness - not eough gene product
Compromised phenotype compared to wt discovered by gene dosage studies.
Define fitness and how it can be measured
Probable genetic contribution of an individual to succeeding generations.
In yeast, relative fitness given by growth rate of different type of cells as they compete for a pool of resources.
Competition experiments give direct measurements on growth rate + fitness in chosen environment
How is heterozygote yeast deletion collection (YDC) used as a model to study dosage effects on fitness?
Uses an available set of oligonucleotide -tagged deletion mutants, each marked by 2 unique 20bp ‘bar codes’
- quantitatively identified using hybridisation array analysis or sequencing
Can monitor impact of gene on fitness in different growth conditions (env effect) by measuirng its up- + down- tag.
Showed haploinsufficiency can be environmentally dependent
yeast - 12-20% genes show haploinsufficiency in minimal nutrient medium
How can YDC be applied to pharmacogenics?
Used in screening to discover molecular drug targets.
Identify yeast losing out in competition assay - that strain carries heterozygote mutation in gene that encodes drug target.
Can screen medically relevant drugs using yeast genome profiling to create library.
Can also identify beneficial mutations in presence of drug - confer resistance
Complete dominance
Homozygote cannot be distinguished from heterozygote at phenotypic level
e.g. phenylketonuria (PKU) is full recessive - only 1 wt needed to carry out function (haplosufficient)
Incomplete dominance
Phenotype of heterozygote is intermediate between the 2 homozygous.
e.g. F1 between white + red -> pink (w/r)
Codominance
Both alleles equally expressed in heterozygote
e.g. IA/IB -> AB blood group
Suppressor mutation
Mutant allele of a gene reverses effect of a mutation in another gene -> allows active protein complex formation despiyte mutations.
Epistasis
Interaction between genes at 2 or more loci, so phenotype differs from expected if the loci were expressed independently
negative/antagonistic: 2-locus epistatic interaction -> smaller combined fitness than sum of their own
positive/synergistic: 2-locus epistatic interaction -> enhances fitness of individual more than sum of their own
overriding mutation is epistatic + overridden one is called hypostatic
Synthetic lethality
2 genes have a synthetic lethal relationship if mutants in either gene are viable but double mutation is lethal
- environementally dependent
How to infer genetic interactions?
- Test single mutants for dominance
- Check if mutants at same allelic locus
- Make double mutants to look for genetic interactions, if 2 genes interact, phenotype different to simple combination of 2 single mutations
Gene interactions between different genes in
biosynthetic pathways
Auxotrophic mutants cannot synthesise cellular components from inorganic nutrients.
