L14-18 Flashcards
why study drosophila malanogaster
- rapid life cycle (10 days)
- easy to obtain mutants
- ~100 years of accumulated knowlage and tools
- inexpensive
- sequenced genome
-drosophila has XY sex determination - small number of chromosomes
what/when is the drosophila malanogaster not undergo meiotic recombination
- chromosome 4
- males
what is a hypomorphic mutation (example)
- partial loss of function
- white apricot mutation causes orange coloured fly eyes
what is an amorphic mutation (example)
- complete loss of function
- white mutation in flies leads to no pigmentation
- notch causes wing abnormalities, needs two copies of wild type for wild type phenotype (dosage effect)
what is an antimorphic mutation
- when duplications are put in there is still not a complete wild type phenotype
- negative activity
- e.g. ebony, interferes with wild type protein function via interaction with mutant protein and wild type protein (implies wild type is a polymer protein
how does an antimorphic mutation work
- anitmorphic alleles act as dominant negatives by competing with and reducing the activity of wild type gene products
- nonsense mutations results in expression of trucated protien, competes with wild type for binding to substrate 1 but cannot bind substrate 2. overexpress a dominant negative muatnt form
what is a hypermorphic mutation
- gain of function
- elipse isgain of function mutation in the drosophila EGF receptor which results in increased singalling, dominant, icreased activity however disrupts eye, gain of function as deficiency restores eye
what types of mutagenesis can occur
- alkylating agent (induces point mutation, very efficient)
- radiation (induces chromosomal rearangement, deletions of varying sizes, inversions, translocations, less efficient mutagen, easier to map location than point mutations)
- pelements (transposons, induce insertion mutaions, low frequency and non random, very easy to map insertion site, large and growing collections of single P-element, insertion lines available
how do alkylating agents work
EMS replaces hydrogen bond in guanine, alkylates oxygen, changes the hydorgen bonding pattern from a 4 bond to a 2 bond and changes the partner to which this base will interact with in the double helix
what is the most used transposon in drosphila
p element
what is the cellular blastoderm cell differentialted into
- pole cells (primordial germ cells)
- 6000 somatic cells
what are the problems of genetic screening
- most mutations are recessive
- many mutations are lethal so you can only recognise the phenotype when the fly is already dead
how to screen recessive lethal mutations and maintain fly stock
- hit male flies with a mutagen
- cross to wild type females (diploid progeny will be heterozygous)
- cross individual f1 males to wild type females
- self cross f2 progeny and identify those carrying lethal mutations (cannot destinguish +/+ from +/-
- to distinguish we use a mutant marker in mutant, i.e. eye/ body colour
what is needed to identify a lethal recessive mutation during recombination
a balancer chromosome
- heavily mutated chromosome from female f1
- chromosome cannot lineup with partner due to structural differences
- supresses genetic recombination
what is an example of a balancer chromosome in action
- a CyO (carries Cy,cn mutations)
- screen will end up with a with a-/CyO, Cy,cn (where a- is lethal)
- crossing them together
- creates two lethal a-/a- and CyO/CyO
- one viable Cy phenotype a-/Cyo,Cy/cn
what are the maternal effect mutations
zygotic mutants
- gap genes
- pair rule genes
- segment polarity genes
- homeotic genes
what is an example of maternal effect gene
bicoid
- female bicoid appears normal (bicoid-/bicoid-)
- male is missing head and thoracic structures (bicoid-/+)
- if some anterior/posterior cytoplasm is replased then tail/head will form
- bicoid gene is from mother
what occurs to the drosophila egg cells during formation
- germ line has 16 eggs but only 1 becomes the oocyte, the rest are nurse cells
- nurse cells form cytoplasmic bridges that feed in mRNA and the bicoid gene
- diffusion of bicoid mRNA is restricted by binding to the cytoskeleton, (causes concentration gradient at anterior end)
- nuclei along A-P exis exposed to varyung bicoid conc.
- bicoid is a transcription factor, able to read position due to diff in conc of biciod
- in humans nucleation in first cell
what are gap gene mutants
several segments of embryo are missing , missing segements are where the GAP genes are expressed
what happens if gap genes are mutated
loss of several segments and regions in the embryo
what is the function of gap genes
- give course, initial sub-devision of the embryo,
- encode transcription factors regulating gene expression,
- give original A-P
- bicoid gradient binds to enhancers that regulate GAP ecpression, different conc enables expression of different gap genes
what are pair rule mutants
- missing alternate segments (either odd or even)
- expressed in narrow stripes in the segment affected by mutation
- encode transcription factor proteins
- integration of singnals regulated by the different gap genes controls expression of pair rule genes
- croeates spatial seperation 4 cells wide
- Second even skipped strip has control regions that respond to certain thresholds of bicoid and other GAP genes called hunchback and a gap gene giant and kruppel, (combine to make a precise even skip)
what are segment polarity mutants
- mutants missing parts of segments
- expressed in narrow stripes down to single cell width
- expression regulated by pair rule genes
- frequently encode signalling pathway components
what mutations occur due to segment polarity mutants
- expressed in a narrow region however are required for the making of the naked cuticle part of the repeating segmental pattern
- mutation of this causes a continously spiky fly
what is the homologous of segment polarity genes in humans
associated with cancer
- cyclopia (fused eye), the region of which hedgehod is expressed disapears
what are homeotic mutations
- do not alter number of segments but alter identity of segments
- are selector genes ( initiate programs of gene expression which conger identity of segments
what is the function of homeotic genes
- evolved from gene duplication
- all encode transcription factors
- contain homeobox domian (a 60 amino acid helix turn helix class DNA binding domain)
- binds to DNA allowing transcription factor activity of the genes
what genes regulate expression of hemeotic genes
gap genes and pair rule genes
- determine position and number of segments and together determine which combinations of hemeatic genes are expressed in what segment
what is a loss of function hemeotic mutation
identity change to more anterior segment
what is a gain of function hemeotic mutation
identity change to more posterior segment
why study the fly eye
- Array of around 800 ommatidia
- Highly ordered packing of ommatidia
- Easily identify mutations that disrupt ordered packing of ommatidia
- Eye is not required for viability of the fly (Maintain stocks of fly that are lacking a functional eye)
how are flies lacking the R7 in the ommatidia identified
do not move to UV light
what are enhancers screens
assumption: if two mutation are in genes within the same pathway then the phenotype resulting from the combination of the mutants will have a greater phenotype than expected from the simple addition of their phenotypes i.e. a synergistic interactio
what are supressor screen
mutation of one gene in a pathway supresses phenotypic connsequences of a mutation in another pathway component
what is a synergistic phenotypic change
when the mutation of two phenotype mutants exceeds the phenotypic reaction two the addition of the two phenotypes
what is an example of conditional mutants
use of temperature sensitive allele of sevenless
- perform screen at temperature where there is just enough sevenless activity to support R7 differentiation, identified 3 genes, Ras, son of sevenless, downstream of receptor kinase
what is RAS
- a GTPase enzyme
- transmits signals from receptors
- has two conformations (GTP signal on and GDP singla off)
- when GTP is hydrolysed it switches to GDP which turns it off
what is the son of sevenless mutation
- functions to activate Ras,
- biochemically shown to act as a GTP/GDP exhange factor
- promotes replacemtn of GTP for GDP
what is downstream of receptor kinase
- SH3 (binds to proline rich protein sequence, in sos
-SH2 (binds phosphorylated tyrosine to sevenless - may be a liker protein linking the receptor to RAS
how is a sepressor screen for negative regulators of a signal tested
- perform screen at temp where not enough sevenless to support R7 differentiation
- identified GTPase activating protein (GAP) (stimulates GTPase activity and promotes conversion of RAS to its inactive GDP form)
what is the human homologe of Ras
an oncogene with a mutation that locks GTP on, so the signal is perminantly on
how does downstream of receptor kinase function
as an adaptor protein between RTK and sos allowing recruitment and activation of Ras
how can epistasis be used to determin gene function in the same pathway and theri function order
- need two mutations with opposite phenotypes
- hypermorphic (GOF) mutation of B (receptor(=) resutlts in increased signal (C) in absence of ligand (A)
what is mosaicism
- dominant mutation in early developement
- could arive through chimerism (cells from a different origin(absorbed cells from a non-edenticle twin that failed to develop)
how does mosaicism arive from transposons
- maize mutations caused by insertion of transposable element
- detected by pigment phenotype
