Lecture 16 Flashcards
i 1. Contrast the approaches of forward genetics and reverse genetics. Which is more amenable to
random mutagenesis? Which is more amenable to CRISPR-Cas9? Explain.
- Forward genetic analysis:
Mutagenize organisms in the lab with unknown phenotype , screen mutants for phenotype identify the causal gene - We begin with a gene sequence, make mutants, and then identify the phenotype, instead of starting by mutagenizing with the phenotype of interest in mind.
Design a forward genetic screen for point mutations that cause cadmium resistance. Choose an
organism, a mutagen, and a method to isolate both dominant and recessive mutations. Justify each of
your choices.
- begin when an inbred ancestor/ clone that can produce 1000s of offspring quickly (yeast, c.elegans, dropshplia) and is crossable
- mutagenize 1000s of these individuals using UVC or EMS to induce point mutations
- look for the phenotype that causes cadmium resistance
- you want to plate cells on cadmium gel
- isolate dominant mutation by crossing a mutant male with a wildtype female as dominant mutation will be seen in the F1 screen
- you want to plate cells on cadmium gel
-isolate recessive in two ways:
- we can use a haploid where refcessive and dom mutation would be seen in one cross
- in diploid organisms, that can self-fertilize(c.elegans), we can cross organisms to themselves which will result in 1/4 OFFSPRING SHOWIN RECESSIVE IN F2
- in diploid organisms that cannot self-fertilize, ( you will only be able to get homozygous recessive in f3 generation)
- you mutagenize male and cross male with nonmutated female to get some F1s that are heterozygous for different mutations
- you back cross the 1 F1 heterozygous mutant with its parents to filter out the non-desired mutations and thus produce 1/4 OFFSPRING RECESSIVE in F3
- since we are trying to screen for point mutations we could tailor the screening to sequence the gene for C to T mutations
Design a forward genetic screen for point mutations that cause increases in susceptibility to heat
induced seizures. Choose an organism, a mutagen, and a method to isolate both dominant and
recessive mutations. Justify each of your choices.
- get an inbred ancestor/ yeast clone that can produce 1000s of offspring
- saturation mutagenesis: mutate 100s of offspring to where a majority of them are mutants
- to induce point mutation use UVC or EMS
- to induce larger-scale mutations use X-ray radiation
-to
Why are F3 screens needed for identifying recessive mutations in organisms that cannot self-but
these mutations can be identified in F2 screens in organisms that can self?
since diploids cannot self cross and the f2 generation will be heterozygous for DIFFERENt mutations you would have to back cross with the parents.
Explain the CLB strategy for finding recessive lethal mutations in Drosophila. Diagram out the three
relevant crosses and indicate which genotypes survive and which die.
a mutagenized male was cross with a heterozygous (CLB) female to determine if mutation was recessive
The clb females are isolated from offspring and crossed to wildtype males
if the mutation is lethal we will have zero males ( hemizygous for mutation and hemixugous for clb chromosome)
if the mutation is not recessi. elethat we have one male as the hemizygous for the mutation will survive and the hemizguous for CLB chromosome will survive
Explain how loss of function in essential genes can be studied in haploids.
by plating mutagenized yeast on a plate a low temperature and then analyzing which colonies die when replica plated at high temperature indicate there is a loss of function so these are conditional mutants
In a forward genetic screen for S. cerevisiae mutants that can grow in elevated concentrations of
azole drugs, you identify a mutant that has moderate levels of resistance but also a severe growth
defect in rich media. How would you go about identifying a secondary mutation that enhances the
resistance phenotype? How would you go about identifying a mutation that rescued wild type growth
in rich media?
an enhancer screen would be used to determine a mutation that enhances the resistance phenotype
or you can uses experimental evolution by allow cells to grow and rich media and over time seeing the cells natural selection
a suppressor screen would allow us to look for mutation that restored the wild type
Describe the two main mechanisms of synthetic lethality. Can three genes display synthetic
lethality? Explain
- two non lethal mutations becoe lethal when together
e.g: a hemizygous male who inhereits recessive prune x from mom and has x ^ prn killer allele will die.
Parallel pathways: if pathway A and b are responsible for an essential biological function. Pathway A can carry on that essential biological function even if pathway B is mutated.
within pathways:
loss of function of one allele results in reduced activity but lethality
loss of function in both alleles result in lethality
Explain how sequencing can be used to identify causal mutations in a forward genetic screen.
you can look for mutations that are present in the f2 but not in the inbred parental. Mutations that are widely shared but not universal due to recombination amongst the f2 will be causal.
What two problems with forward genetic screens does experimental evolution circumvent?
Mutagenesis drawbacks:
1. Biased mutation spectrum:
whatever mutagen used will have a biased mutagen spectrum and can only cause certain mutations
- ecological relevance of the mutants recovered from mutagenesis can be questionable
antagonistic pleitrophy: mutation may give desired phenotype but it can be associated with a low fitness phenotype
who does experimental evolution solve this?
the organisms grown in the lab will have deliberate selective pressures which will allow the natural instead of biased spectrum of mutations to be used and low fitness mutations will be selected against
What is the function of Cas9? tracrRNA? crRNA?
The CRISPR-Cas system is a defense mechanism against invading nucleic acids
crRNA’s are subunits of transcribed palindromic repeated that have identical repeat sequence and a unique spacer of phage DNA
tracrRNA’s can base pair to crRNA and can bind crRNAs to cas9
When bacteria has a phage spacer in its crRNA that is complementary to the invading phage RNA, the base pairing allows cas to make a double-strand break and destroy the phage infection
The original function of CRISPR was expanded such that:
the system was simplified to fuse the tracrRNA and crRNA to function in one molecule known as guide RNA
you can also provide a synthetic guide RNA template to the cell that will guide CAS9 to a complementary gene of interest that needs to be destroyed.
You can also do site-directed mutagenesis where the double stranded breaks are repaired by Homologous recombination or SDSA to introduce a single nucleotide change.
If a bacterium survives a phage infection, what is a likely outcome for its CRISPR array? Is this
change heritable?
new spacers (seqs derived from phage) can be inserted into the CRISPR array as you survive a page infection. So as the microbe continuously survives phage it will cleave the out the infections sequences it can be passed to offspring
You have a stock of wild type C. elegans. Suppose you want to assay the effects of loss of
function of a particular gene in this stock. Propose an experiment to accomplish this without altering
the genotype of your wild type stock.
you could use RNAi to knock down the gene of interest by injecting dsRNA that is complementary to the gene of interest into ecoli. When the c.elegans eats the e.coli , the dsrna is processed by dicer and then be associated with RISC which degrades the target mRNA leasing to zero gene expression.
You are studying a gene in mice that is important for proper formation of the brain, and this gene
is only present in the brain in the wild type. You construct two chimeric alleles, one with the gene
under the control of a promoter that causes this gene to be expressed throughout the body later in
development, and another that causes high expression in most areas of the early embryo. The first
gene yields viable mice, but upon inspection you find that the concentration of a cell type called
astrocytes in the spinal cord has increased dramatically. The second allele leads to early embryonic
developmental arrest and death. What, if anything, can you infer from each of these alleles?
too high of expression in early development can be lethal as there is early embryonic death.
We can infer that the gene being under this new promoter can lead to the development of astrocytes
