End Flashcards
(21 cards)
Three different ways to identify genes
Make a library of cDNA clones from mRNA.
Make a library of genomic clones and making predictions based on genomic sequences.
Microarray
Reverse genetics evaluate
What does it answer
Time consuming and expensive.
Powerful and effective.
First you clone the gene.
Then you either do gene replacement or gene knockout.
Replacement is where you alter the gene and don’t destroy it.
Knock out completely removes the gene to help determine its function.
Test whether a gene is essential for embryonic development because if it is the progeny will not survive when it is knocked out.
Gene knockout in reverse genetics
First get a clone of the gene.
Insert two non mammalian genes.
Put NEO into an exon to destroy the activity of the gene.
And TK is inserted off to one side.
The exon containing NEO is called the homologous arms because it is homologous to the mouses genome.
The modified gene is then put into mice embryonic stem cells.
The cells DNA repair mechanisms recombine the gene into the mouse genome.
This is ineffective and rarely works.
If the integration of the new gene is done by homologous recombination it causes TK to be lost. This isn’t common.
Double selection to get the cells we want.
Distinguishes between cells with genes added by homologous recombination and cells without.
All the cells are grown on neomyocin.
Cells that don’t contain NEO will die so only cells that took up the new gene will survive.
The survivors are exposed to GANC to kill all the cells containing both NEO and TK.
This will only leave the cells that took up the gene by homologous recombination and is called negative selection.
The cells remaining will have a knockout of the gene due to NEO.
After you have the cells with the knock out.
Put into mice embryos.
Embryo put into a mouse.
The mouse will give birth to transgenic progeny.
The first generation will be mosaic and will have a mix of inserted cells and normal cells from the original embryo.
They have Mosaic gonads and are bred to make non mosaic carrying progeny.
Then they are bred to make homozygous mutants for the knockout.
And from this you can look at the mutants for phenotypes and work out the function of the gene.
Foreword genetics
Identifies genes and finds their function.
It is done by randomly mutating the genome rather than choosing a gene like in reverse genetics.
Look for interesting phenotypes in the offspring and identify the genes that cause the defect.
Animals used for foreword genetics
Random mutagenesis affects the whole genome so we have to analyse many animals.
Yeast, C elegans, drosophila, zebrafish
Screens can be done for dominant and recessive traits.
Foreword vs reverse
Foreword genetics starts with the phenotype and you find the genetic code.
Reverse genetics starts with the genetic code and you find the phenotype and function.
Positional cloning
Use a genetic map to find a mutation and identify the gene.
Foreword genetics in fly’s
Why are men used
Male flys are treated with EMS mutagens. Males are used because sperm is easier and quicker to make than oocytes.
The starting parent fly’s are PO. A wild female and a hetero mutant male.
In the male every sperm will have different mutations so each of the F1 progeny will be unique and have different mutations.
A single F1 male is mated with a wild female to make F2. All the F2 will have the same mutations that were passed from F1. 25% will be carriers of the mutation.
The F2 carriers are bred together to make a homo mutant for the gene F3 which is 25% of F3. The phenotype is now visible.
Punnets for each step
How many generations
The F1 will be 50% wild and 50% hetero.
The F2 will be the same.
The F3 will be 25% wild. 50% hetero and 25% homo if it’s recessive.
It takes three generations to make a homo mutant for recessive.
Two mutations giving the same phenotype.
If two separate families of F2 are showing the same phenotype, are the mutations on the same gene or not ?
Breed one of each of the families together.
If they are different alleles of the same gene, 25% of the offspring gives a mutant phenotype.
m1,+. And. m2,+. Gives m1,m2.
They are not complementary.
If you breed them together and there is no phenotype then it means they are on separate genes and they complement eachother.
There must be more than one gene that controls that phenotype.
M1,+ and m2,+ gives m1,+ and m2,+
Complementary analysis pictures.
If they are on the same gene the will be opposite eachother on the chromosome.
If they are on different genes they will be at different levels in the chromosome.
Miss sense and non sense
Miss sense- alter an amino acid in the sequence
Non- introduce an early stop codon
Amorphic
Missense mutation that completely inactivates the DNA binding domain, non functioning.
Hetero means there is enough healthy protein to allow things to function correctly, only one allele is mutated so 50% of proteins are okay.
Haplosufficient. Only for recessive.
Homo leads to 100% faulty protein and no transcription can occur as all the binding proteins can’t function leading to lethal phenotype.
Hypomorphic
Missense mutation that weakens the DNA binding domain.
Hetero means the binding domain can still bind but falls off the DNA easier.
Half of the proteins will be healthy so no phenotype will be shown.
Homo means all proteins are weakened and they always fall off frequently leading to less transcription.
This causes a milder phenotype than the amorphic mutation.
RECESSIVE
Antimorphic
Missense mutation that destroys the dimerisation domain.
A dominant mutation.
The DNA protein can bind to the DNA but it cannot dimerise because the dimerising protein is bad.
Hetero will not be able to dimerise so won’t undergo a conformational change to become active.
Transcription can only occur when two wild types dimerise. So this gives a weak phenotype as 50% are okay.
Homo will completely inactivate transcription.
Hypermorphic
Missense mutation that results in activation that is independent of dimerisation.
Hetero means the protein is activated all the time so doesn’t need dimerisation to cause a conformational change.
This will increase transcription and make too much mRNA.
Homo will do the same but worse because it is 100% or proteins that are active all the time.
Antimorphic as an inhibitor
It is a competitive inhibitor.
Called dominant negative.
The mutant protein poisons the wild ones.
In hetero the mutant will stop the healthy from working.
How to study genes whose knock outs would usually kill the embryo
Generate conditional knockouts.
You can choose where and when it will happen.
Add loxp sites to the start and end of the gene.
The mice will develop normally.
Cross this mouse with one that contains a tissue specific cre recombinase gene.
This will allow deletion of the gene between the loxp sites but only in a specific tissue so the mice won’t die.
Speman and mangold 1924
Transplanted a region from the dorsal side of one embryo and put it on the ventral side of another embryo.
It developed and had axis duplication because the region transplanted was the organiser.
