L 9-13 Flashcards
what is an example of protein isoforms
notch in mammals
- proteins that are similar to each other and perform similar roles within cells
what are the main issues found with forward genetics
- production and mapping of mutants is a long process, using tools exclusive to yeast and flies
- pleiotropic genes have multiple function, can generate lethal phnotypes (need to use mosaic or temperature sensitive conditional systems)
-redundancy amongst genes in the same family or paralogs
what is loss of function
- production of no or less proteins or a protein with reduced or no activity
what is gain of function
- production of more proteins or an extended expression pattern (new time and/or new place), or a protein with an increased activity or new function
what are the three types of loss of function
- amorph
- hypomorph
- antimorph
what are the three types of gain of function
- hypermorph
- neomorph
what is the LOF and GOF effect on transcription
- amorph
none - hypomorph
reduced - antimorph
normal - hypermorph
increased - neomorph
extopic
what is the effect of the LOF GOF
- amorph
none - hypomorph
reduced - antimorph
antagonistic - hypermorph
increased - neomorph
novel
what is the LOF GOF of translation
- amorph
none - hypomorph
reduced - antimorph
normal - hypermorph
increased - neomorph
normal
what is the LOF GOF for function
- amorph
none - hypomorph
reduced - antimorph
reduced and antagonises wild type - hypermorph
increased - neomorph
new
what is the LOF GOF often
- amorph
recessive - hypomorph
recessive - antimorph
dominant - hypermorph
dominant - neomorph
dominant
describe an amorph
- often recessive (unless on the X chromosome) due to the normal allele compensating for the loss
- when they are dominant, they occur in haplo insufficient genes
- haploinsufficiency usually occurs when the protein is in a protein complec or if the quantity is crucial
describe a hypomorph
- often recessive (unless on the X chromosome) due to the normal allele compensating for the loss
- when they are dominant, they occur in haplo insufficient genes
- haploinsufficiency usually occurs when the protein is in a protein complec or if the quantity is crucial
describe an antimorph
- dominant negative
- interfere with normal gene function - more severe than one copy of the amorph (null)
common with proteins who have binding partner (dimer, receptor), inhibits the partners function
describe a hypermorph
- high transcription or failure to degrade transcript or protein
- sometimes constitutively active (a receptor is normal activated apon ligand binding. if mutation is on binding site, then independent of ligand and alsways active)
describbe neomorphs
- novel function for protein arising from mutation (new catalytic activity/binding partner)
- commonly due to change in expression pattern (in regulatory regions controlling expression)
- can also arise from translocation from chimeric fusion proteins
what types of chemical mutations can be generated for forward genetics
chemical
- ethylmethane sulfonate (point mutation) flies. worms, plants
- nitrosourea (point mutation) mice
what type pf radiation mutations can be generated for forward genetics
radiation
- uv light (small mutations) many
- x rays or gamma rays (chromosomal rearagnements and deletions) many
what type of insertion mutagenesis using transposable elements can be used for forward genetics
insertion mutations
- transposable elements (disrupt gene/promoter or small deletions) flies, worms, plants
- retrovirus (disrupt gene/promoter or small deletion) mice
what is the downside of mapping mutants through chemical and radioation mutagenesis
- takes a long time, can be combined with NGS for faster results
what is a transposon
- mobile DNA element, jumping gene (make ~50% of human genome
contains - 2 inverted repeats, at start and end position
- gene coding for transposase including promoter, used for cutting and pasting transposon
what are the steps of a transposon
- The transposase acts as a restriction endonuclease and an integrase.
- Step 1: Once expressed, the transposase binds the inverted repeats (IR) and cuts out the transposon (EXCISION), removing some nearby sequences in the process.
- Step 2: The transposase (bound to the transposon) cuts the host DNA like a restriction endonuclease.
- Most transposases bind very small specific target sequences which are found frequently, so creates seemingly random mutation
- Step 3: The transposase integrates the transposon within the cut made in the host genome.
what happens if a transposon is inserted into an exon
- might distupt protein function
- if it is not in a number of three then the open reading frame will be messed up
- effect depends on exon size
if a trasposon is inserted into an intron
- may modify bingin site of the splicing factor, may prevent the spcing from taking place, intron in the reading frame
if a trasposon is inserted infront of a gene
may effect reggulation of a gene due to trascription genes before gene
what should be in a COI (construct of interest)
splice site
marker for integration
reporter gene
who discovered transposons
barbara mcclintock 1940s, nobel prize 1983
what diseases are trasposable elements ivolved in
- Neurodevelopmental disorder
- Myocardial infarction
- Modulation of immune system
- Neurodegenerative diseases
what are the two steps of mapping
- use markers to find out on which chromosome your mutationis: association mapping
- use markers to find out where on the chromosome your mutation is: linkage mapping
what are the two types of markers
- phenotypic markers
- molecular markers
what are phenotypic markers
- Alleles with obvious phenotypes - for years, with geneticists only relied on these marker and flies were the model organism of choice
what are molecular markers
- SSLP, simple sequence length repeat polymorphism, microsatallite
- SNP
what are the limitations of traditional mutagenesis screens
- Traditional screens focus on genes that affect specific phenotypes
- fail to identify genes whose functions overlap or are limited to specific phases of development, or genes that are expressed in a limited number of tissues.
- Genome projects have revealed that traditional screens have assigned functions to only a minority of a given organism’s genes.
- Can investigate gene function through an investigation of expression patterns.
what is an enhancer trap
- TE insertion allows the detection fo the expression controlled by the nearby enhancer elements
- is called an enhancer trap as the COI is a reporter gene and a mutant insertion (we only care about the insertion near enhancer elements)
how do we identify the gene present
- sequence the transposable element flanking DNA
- if the enhancer element is close to the gene, you can identify a gene of interest
- 98% of the fly ET, the TE insertion doesnt modify endogenous gene expression
what is required in the COI for reverse genetics
- Promotor
- Marker of integration
- Normal gene
- Mutant gene inserted randomly and then see what happens
- Sequence we know is required for reverse genetics
- Still inserted randomly in the genome
- Adding a construct into the genome
principles of targeted insertion through homologous recombination (HR)
- COI surrounded by sequences homologous to targeted insertion site
- COI delivered as a linear fragment or a vector
- HR occurs in meiosis and before each mitosis
- one part of the endogenous gene is replaced by one part of the vector (target insertion)
what sequences must be known for the insertion to work
- we need to know BC, DE for the crossing over to work
- we need to know exactly where we want it
- 2.5-6 kb of flanking homologous sequences required
What is the benefit of targeted insertion
We do not have to wait for the mutant, we know exactly where it is
what is a knock out
- delete a gene/promoter or a critical portion of it
mutant - marker replaces all or a great part of the endogenous gene/ promoter
what it knock down
- delete a small portion of a gene/promoter
mutant - marker replaces a small part of a gene/promoter
what is a knock in
- add contruct to genome
mutant - DNA (specific mutation, reporter gene, extra gene from same or other species) often fused to marker
what is needed for a knock out
2 integration markers
- one to replace your gene or gene portion or promoter
- one to ensure the integration happened via HR
how to do a knock in
- replace exon X with a reporter gene using the KO method, select cells with reporter gene and apply drug , cells without M2 survive as M1 is anibiotic
- replace the reporter gene with your mutant DNA and apply drug, cells without M2 survive
what amorphs result from Kock out mutations
targeted deletion in gene/promoter
what hypomorph result from knock down/ knock in mutation
targeted small deletion in gene/P or introduction of key mutation
what anitmorphs result from knock in mutations
targeted small deletion or introduction of key mutations
what hypermorph (overexpression) result from knock in mutations
bypass or boost promoter replace gene by mutant with increased translation or a consitutevely atived allele
what neomorphs (misexpression) result from knock in mutations
place gene under the control of a new promoter or introduce a mutant that changes function
how are knock out’s done with crispr
Non homologous end joining
KO with 1 cut (indel) or 2 cuts (large deletion)
- repairs are very rarely in multiples of three
- easy production of amorphs
how are knock in’s done with crispr
homologous directed repair
easier than HR because requires much shorter homologous sequences (can do 2 cuts for large insert
what are the two types of HDR
short range HDR
long range HDR
what is short range HDR
- most efficient but only inserts small fragments (GFP)
- use Cpf1 instead of cas9
- doesnt do blunt ends when it cuts rather sticky ends
- different repair mechanisms
- allows very small construct to be inserted into the genome
what is long range HDR
- same sa HR mechanism but more efficient because cut pushes repair to occur, therefore needs less homology sequence to work
- more effective than HR
what is the size of insert with short range HDR
insert 10bp-2kp, 30-40 bp of homology
what is the size of insert with long range HDR
insert: up to 6kb, 500-1500bp of homology
normal HR: 2.5-6kb of homology
why is ca9 not used in HDR
p53-mediated DNA damage response
- uses Cpf1
- p53 prevents cancer
what are the cons of using CRISPR-cas9 in making mutants
- off target effect
- can be toxic to cells (p53-medaited apoptosis of p53 mutated
- edits are perminent (not always desired
what is the use of CRISPR-dead-Cas9
regulates transcription
- dead Cas9 can be fused to another protein. it binds specifically to DNA where it delivers the fused protein
- can be fused with a transcription repressor or activator domain
- can also be transcripted with epigentic modifications such as dimthylase and methylase
what is CRISPR KO screening
look at a phenotypic effect of treatment when a gene is KO (amorph)
-uses cas9
what is CRISPRi screening
interference screening (knock down - hypo) - look at effect on essential genes
- Dead cas9 fusion
what it CRISPRa screening
activation screening (hyper) - look at effect when gene is overexpressed
what is the driver line
specific complementary DNA
what is an example of sanctio-temporal iduction
tet on/off
how does tet-off work
addition of a drug (tetracycline or doxycycline), inhibits binding of tTa to TRE (target transgene) causing the gene expresseion not to occor
how does tet-on work
reverse tetracycline transactivator (rtTA) alone does not bind to TRE meaning no activation
however addition of rtTA with a drug such as doxycycline causies transcription
what are the two systems of TET
TET technology comprises two complementary circuits: the tTA-dependent circuit (TET-Off system) and the rtTA-dependent circuit (TET-On system).
when is RNAi typically used
to make amorphs or hypomophs (only affecting transcription)
how does RNAi work
blocking or reducing the expression of chosen gene (knockout or knockdown)
what is the prefered organism for RNAi
worms (not amenable to other genetic approaches)
what are the steps for RNAi functio
- dsRNA is longer than needed
- Recruits dicer
- Dicer makes it a 22 nucleotide system
- System finds complementary RNA
- Binds to and degrades it
how is RNAi delivered
- Synthetic molecule or in inducible plasmid (by drug/nutrient) - short lived action.
- In a virus that can insert in the genome (only way to create stable RNAi lines). Choice of virus: infect all or specific cells
- Worms are given the dsRNA in the bacteria they feed on.
- For plants, the dsRNA is often applied to a cell culture or through a virus that can insert in the genome.
- For flies, the dsRNA can be injected in the developing embryo or inserted and made inducible via the UAS-Gal4 system
- For mice, the dsRNA is introduced in embryonic stem cells using viruses and made inducible via the Cre-loxP system/ Tet On/Off system
what are the pros of RNAi
- In theory should knockdown ALL the normal copies of the gene (i.e. create a homozygous effect) particularly interesting for polyploid plants and for non model organisms whose genome has been sequenced (e.g. mosquitoes, trypanosome, etc)
- Worms are very good at showing the RNAi effect easily in all cells
what are the cons of RNAi
- Effects are variable for different genes and different organisms. Worms are very good at showing the RNAi effect easily in all cells, but some organisms only show it a subset of cells.
- RNAi shows sometimes cross-reactivity and an off target effect - i.e. knockdown other RNAs with similar sequences.
- RNAi does not work in yeast
