week 7 Flashcards
Forward Genetics
What genes are important for a process? Phenotype to genotype
Reverse genetics
What process is the gene important for?
genotype to phenotype
40 years of reverse genetics
if I remove the gene what happens to the phenotype
Testing Forward Genetics
Uses mutagenesis to generate a random pool of genome variants
performing a selection or a screen
Selection
Identify genes that are important for the effect; only resistant variant survives
Screen
Look through all the variants for the phenotype, all the variants survive
Genetic screens
genetic analysis requires genetic variants
to dissect the biological process, apply genetic analysis
Saturation screens
An attempt to identify as many genes whose products contribute to the process that you are studying as is statistically and technically possible. Only generating in all of these methods a random pool of DNA sequence genetic variants
Genetic screen for leucine auxotrophic yeast
looking for genes that are important for synthesis of an important component
mutagenesis of haploid yeast cells, each cells have random DNA sequence changes, we don’t know what the effect of the changes are, look for colonies that reuqires leucine to grow, implying a mutation has inhibited its ability to grow on plates where leucine is absent.
If you have 100 leu auxotrophic yeast mutants does that mean there are 100 genes regulating leu?
No. You can have multiple independent alleles in the same gene.
That encodes for components required for the synthesis of leucine. One gene can get his five times at different positions in the gene
Complementation analysis
Non-complementation in same complementation group=same gene
Independent mutations in the same gene, if you cross the haploids to create a diploid, this diploid will still lack the ability to synthesis leu, non-complementation
Complementation
These two loci have wt alleles and can grow on media without leu; mutations on two different genes
Haploid genetics vs diploid genetics
Haploid: only one gene knockout is required for the phenotype
Diploid: need two knockouts to get to the phenotype
Haploid
Single generation
Large number of variants to screen or select (10^6-10^8)
Diploid
Several generations: we need to perform crosses to get recessive homozygous cells that will show the phenotypes
Small number of variants (10^3-10^4)
Maternal vs Zygotic genomes
early phase: information coming from the mother; zygotic genome is not transcribed for hours/days until after fertilization
oocytes doesn’t go onto meiosis until later
the reason for this is that in animals the zygotic genome is not transcribed immediately. So all the early steps have to be run off of the genetic material in the egg which was provided by the mother
Zygotic
when we’re thinking about genes expressed from the zygotic genome, to screen for those activities that are important, all we require is a male and a female that carry the mutational change.
that will create a zygote, will be homozygous for that mutational change, and if this gene which has this DNA sequence variant, inactivates it, is important for a process, then you will see a phenotype in the developing zygote.
Maternal affect
Transcripts stored in the egg are translated and produced early in the genome.
Now these transcripts are then placed into the egg and after the egg is formed and laid and fertilized, these transcripts that have been stored in the eggs are now translated, producing the product early during development, that allows the egg to develop without transcription of the genetic information of the zygotic genome.
Maternal effect mutants
In this case the mother that is homozygous for bicoid loss of fucntion allele lays a normal egg, but every single one of her egg that she ;ays the larvae will develop wihtout a head 100% of the time, so this information that is required to be passed from the mother to her progeny in this case is gone and now because of that missing infromation the ehad doesn’t develop
Zygotic screens
3 generation screen
Mutagenize the sperm to generate F1 generation
F1 generation contains one sequence variation change in our gene interest (possibly)
cross with an un-mutagenized individual to generate males and females heterozygous for the seuqence change (F2)
cross the heterozygotes to generate 1/4 of individual homozygous for a sequence change
is the embryo dead or did the mutation not have an effect and we screen for things that are important embryogenesis.
Maternal effect screens
we mutagenize sperm, have an individual
which we cross to have more individuals; males and females that are heterozygous. we crossed them to create a mother F3 that is homozygous, that her maternal genome is homozygous. She will be normal looking but when she lays an egg, which would be the F4 generation, those eggs will not develop.
Mutational tagging
DNA sequence variation tgs the gene that you have identified as important
genome sequencing has made this easier.
Reverse genetics requires
reintroducing DNA into an organism defines it as a complex refined model organism.
whats the gene important for, make a mutation of the gene and reintroduce an altered copy of the gene into the organism
Attribute of good model organism
ability to reintroduce DNA
Four ways to reintroduce DNA
Transformation
Injecting DNA
Transposon/viral mediated transformation
Site specific recombination
Transformation
Episome (plasmid; can freely replicate in the organism)
Random insertion
Homolgous recombination
Requirement for transformation
first treat cells to make them competent to take up DNA
yeasts; tissue culture cells
Episome
dominant selectable marker
origin of replication
once in the organism they can freely replicate as an epichromosome, one that is not part of the genome
Episome
dominant selectable marker
origin of replication
once in the organism they can freely replicate as an epichromosome, one that is not part of the genome
Selectable marker
in bacteria it codes for resistance to a selectable marker
in yeast the plasmid will contain a biosynthetic pathway functional marker, Leu2 functional allele, in the chromosome (endogenous locus) the yeast contains a loss of functional allele. This results in the cell requiring leucine for growth
Random insertion
occurs in tissue culture cells when they are being transformed with DNA, the DNA is able to be inserted in a eukaryotic chromsome and once its inserted it will be stably maintained generation upon generation. This insertion is random, an not necessarily at the locus of DNA we are introducing, random insertion may lead to lack of gene expression
Homologous recombination
Our gene in the chromosome has DNA introduced using transformation, this DNA contains a region homologous to the gene and a dominant selectable marker. This DNA will rarely undergo homologous recombination
the marker is incorporated into the endogenous locus and will disrupt the gene
targetting your transformation event to a particular locus in the cell
Injecting DNA
Very large cells (vertebrate, nematodes and insects)
a needle that contains the DNA that you insert into the cell and pump in the DNA
generates random insertion
Transposon/viral mediated transformation
In some cases, transformation/injection of DNA does not result in reintroduction of DNA.
It needs help sometimes
Transposon mediated transformation
transposases will bind to the inverted repeats + transposases coding region
copy+paste mechanism
Transposon mediated transformation
transposases will bind to the inverted repeats + transposases coding region
copy+paste mechanism
Transposon mediated transformation (two compotents)
transposase: the trans-acting factor
inverted repeats: cis acting factor
seperate the components; we need to control when transposition occurs
inject one plasmid that contains transposase but does contain inverted repeats; and another plasmid that contains the marker and whateber gene we are trying to reintroduce flanked by inverted repeats to which transposase will bind.
random insertion
Transposon mediated transformation (two compotents)
transposase: the trans-acting factor
inverted repeats: cis acting factor
seperate the components; we need to control when transposition occurs
inject one plasmid that contains transposase but does contain inverted repeats; and another plasmid that contains the marker and whateber gene we are trying to reintroduce flanked by inverted repeats to which transposase will bind.
random insertion
Viral mediated transformation
incorporate your gene and a marker into a viral genome, take advantage of cirus that integrate their genetic material into the hosts genome.
Take the viral genomes and incorporate your gene and marker into the host genome
package the genome into a viral particle
After viral infection your gene is inserted into the genome of the cell.
Site specific recombination
recombination sites are recognized by recombinase, recombinase mediates recombination between the two sites.
place a recombination site in the genome, and add DNA in the plasmid that also contains a recombination site + GOI+ marker, express recombinase in these cells as well
Use of transformation/transgenesis
Functional complementation
Misexpression and gain-of-function
Knocking out genes
Functional complementation
In a forward genetic screen we have identified mutations in genes important for a process
what are the genes associated with the mutations
phenotype to gene
What are the genes associated with the mutation, what is the normal function of the gene
Functional complementation steps
put random pieces of DNA in the plasmid and replicate them in yeast
1-extract DNA from wt yeast; all loci have functional alleles
2-fragment the genome with restriction enzyme
3-take random fragments from the genome and clone them into yeast plasmids
4-create a library of random fragments form the WT yeast genome
5-yeast with non functional cdc24 gene
6-transform each one of the episomes into the yeast and look for phenotype (functional cdc24)
7-extract the plasmid and make mutations in cdc24 gene
8-What part of the cdc42 gene are important for function, do this by making mutation, ask which mutation do and do not complement lf
Map out functional important regions
Misexpression and gf
What happens when we mis-express the gene, do we see an alteration the phenotype. Do a designed approach
Antennapedia is expressed in cells that will give rise to the second leg.
What if we misexpressed antennapedia in the antenna cells of the organism
Transposon mediated transformation of antennapedia
marker, hsp and antp cds
heat shock promoter results in heat shock inducible ANTP protein expression, results in ANTP expressed in all cells
make a very specific construct
Mis-expression results in antenna to leg transformation
Knocking out genes
Knock out genes via homologous recombination
great way to test the function of genes
Homologous recombination
DNA molecule that contains a marker is introduced into the cell, introduce that into the cell and this DNA will search through the genome looking for homologous DNA sequences and it it finds it the homologous recombination event will occur.
Dominant selectable marker
the marker selects for homologous recombination
the insertion of the marker into the gene is the mutational event that disrupts the gene
the marker is inserted into the gene via hr
we need a very strong selection in order to identify cells where this event has occured.
the marker is the mutaitonal event that disrupts the gene
Systematic gene knockouts in yeast
functional genomic, all yeast genes have been knocked out and you can screen strains for which gene is required for your process
you can screen these strains for which gene is required for your process
Strain one is missing a gene.
Screen each strain for a gene required for your process.
RNAi
Double stranded RNA complementary to mRNA
Inhibits mRNA by degrading it, the gene is unaffected
no mRNA
no protein
Mechanism of RNAi
Double stranded RNA is digested by an RNAse called dicer
fragments are recognized by ago and with other factors creates a RISC. It is target to our specific mRNA because of the ability of the RNA fragment to bp with mRNA
Major Points of RNAi
Specific mRNAs are targeted for degredation because of the complementarity of the RNA bound to AGO and the mRNA
The RNAi mechanism is thought to have evolved as a mechanism that supresses parasitic genetic elements: viruses, transposable elements
miRNA and soil worm development
speical future of development is its invariant lineage it goes through a stereotypic series of cell divisions: We know at specific times what cell will be present in the developing embryo or larvae.
each one these divisions can be followed by using microscopy because it is a transparent organism
lin-14
Timing is affected, allows us to identify mutants.
Lf no complicated first lineage, l2 lineage is occurring during L1.
Gf leads to a complicated L1 lineage which is repeated again.
lin-14 encodes a nuclear protein
lin-4
This gene is important for supressing the L1 lineage.
lin-4 encodes a non-coding RNA a micro-RNA-the first found
Lin-14 gf
lin-4 rna base pairs with the lin-14 3’ utr
lin-14/lin-4 duplex 2 (bulged)
in lin-14gf mutants the lin-4 binding sites are deleted
lin-14/lin-4 mRNA/Protein expression
Expressed at all stages of larvae development but is not active during L2
protein expression falls during L2
lin4 miRNA expression starts at the L2 stage and remains present at all stages, as it accumulates and binds to the mRNA it suppresses the translation of lin14 mRNA
miRNA mechanism
miRNA is apart of your genome, they are transcribed genes (RNAPII) produces a primary miRNA transcript that is inactive, it is processed to a pre-miRNA and exported from the nucelus where it is processed by dicer
dicer slices it into small pieces and AGO binds to the miRNA
recognized by cofactors to form the RISC
Argonaute protein family
Multiple proteins encoded in the genome
AGO1 miRNA silencing
AGO2 RNAi mRNA cleavage
8 AGO proteins have different biochemical properties
miRNA vs RNAi
miRNA surpresses translation of mRNA
RNAi degrades the mRNA
No protein is produced by distinct mechanism
miRNA role in RISC
it guides the RISC to specific mRNA
it does not activate the RISC proteins
If you could get AGO onto the mRNA idenpendently of the miRNA, the RISC proteins should still work
tethering ago to lambda n RNA binding protein still allows for RNA silencing
Functional genomics with RNAi
Ecoli expresses a double stranded RNA, nematodes eat ecoli and absorb the double stranded RNA
Double stranded RNA is getting to all the cells and inhibting gene products
Ecoli nematode plasmids
cDNA is inserted between two promoters; to get double stranded RNA
20,000 strains with a cDNA for every gene, into each ecoli.
transfect 20,000 nematode with one ecoli each to knock out every single gene and look at the phenotype
nematode eats ecoli and absorbs dsRNA, that silences the gene of interest
Defenses against genomes
1-RNAi/miRNA
2-Restriction modification
3-CRISPR
natural defenses against genomes, selfish elements, viral DNA
Bacteriophage
Bacteriophage infects the bacteria
viral DNA takes over the host machinery replicates its RNA and is packaged into viral shells
The bacteria lysis open and the phage viruses are released
Restriction modification immunity
Source of restriction enzymes
Origin of recombinant DNA technology
- RE sees phage DNA, binds to recognition site and chops it up into little pieces
- methylase methylates all the genomic DNA restriction sites so that the genomic DNA is protected from the action of the restriction enzymes
CRISPR mediated immunity
1-Acquire
2-Immunity
3-Reeingineered
acquire information from invading DNA store it in its genome and use it to defend itself
CRISPR Components
Clustered regularly interspaced short palindromic repeats
cas: CRISPR associated
PAM: protospacer adjacent motif
Bacterial CRISPR Steps
1-Cas enzymes are able to cut off a section of the invadgin DNA next PAM, cas binds to PAM and then cuts the DNA
2-Cas binds the piece of viral RNA and inserts it into the genomic DNA at a CRISPR array
3-When a phage infects again, pre-cr RNA is transcribed
4-pre-cr RNA is processed to release each spacer which is homologous to past invading viruses
5-Tracer RNA binds to a motif adjacent to the cr-RNA spacer
6-Tracer RNA allows for Cas9 to bind
7-Complex formation, Cas9 protein with guide RNA will find the PAM in the phage genome and then unwind the DNA using guide RNA as a template and then induce a double stranded break in phage genome inactivating it
Why doesn’t cas9 cleave the genome
no PAM in genome, a sequence called PAM is just NGG, that is next door to the DNA that is going to cut out
highly unlikely that the genome is cleaved, 44 bits of information is very high specificity
Reeingeenred CRISPR
Make RNA which contains the recognition seqeunce and the tracer RNA into a chimeric RNA
Two component system
Double stranded breaks
Needs to be repaired:
End joining
homologous recombination
DSB are very dangerous, they initiate mitotic recombination
End-end Joining
NHEJ, enzymes reseal the break, they are not very precise and in the process of repairing the break introduce indel mutations that can affect gene functions.
Homologous recombination
DNA contains a selectable marker that has homologous arms to the gene you want to insert your selectable marker in to create a mutation/bring in a new DNA element
Enzymes are brought to the spot to induce recombination
Mutation of Cas9
Introduce mutation in the areas that make the double stranded break, they cannot make cuts
because of the high information content of its binding you can locate unique sequences in the genome
Inhibitor of transcription
Activator of transcription
Florescence protein
