Chapter 7 Flashcards
Manipulating genes in vivo
1) Random mutagenesis of the genome = Cells can be treated with low doses of chemical or radiation to induce mutation of genome
2) Gene deletion = Any gene (or region of DNA) can be precisely deleted, effectively eliminating gene function
3) Gene replacement = delete gene and replace with of mutant of the gene
4) Cloning in vivo = Homologous recombination can be used by the cells to ‘clone’ DNA or recover a mutation in the genome
Whole genome mutagenesis strategies
1) Chemical treatment = Ethylmethane sulfonate (EMS) or methylmethane sulfonate (MMS) adds alkyl groups to bases. Subsequent rounds of replication result in the incorporation of incorrect nucleotides across from the alkylated bases
2) UV radiation treatment = Low doses of UV lead to pyrimidine dimers that alter the structure of the DNA strand. Upon replication, incorrect nucleotides can be incorporated, leading to mutiation
Non-homologous end joining (NHEJ)
This is the predominant mechanism for ‘fixing’ broken DNA in higher eukaryotic cells.
The basic idea: DNA breaks are rejoined to form an intact DNA molecule
Problem: This is not a high fidelity mechanism
Reason: DNA ends are processed in such a way that bases are often added or lost (usually lost)
The result: DNA sequence has been altered, and there is the potential for gene mutation (disruption)
Homologous recombination (HR)
This is the predominant mechanism for ‘fixing’ broken DNA is yeast (lower eukaryotes)
The basic idea: DNA breaks are fixed by ‘copying’ information from a homologous chromosome
The result: There is no insertion/loss of genetic information; however, genetic information may be changed
Advantages for us: We can insert DNA into the cell, and HR can incorporate this information into the chromosome.
Gene deletion by one-step gene replacement
Requirements:
1. A
selectable
marker
to
amplify(e.g.,
kanMX4)
2. Primers in
which
the
5’
ends
contain a
stretch
of
40‐100
bases
of
homologous
sequence.
The following outcomes are possible:
1. Unsuccessful deletion, ORF still present (will not grow on media containing G418 antibiotic)
2. successful deletion, kanMX4 module replaces ORF (Cells can be selected for by growth on G418 antibiotic
Gene deletion of essential genes in yeast
Reminder: yeast can exist as a haploid cell or as a diploid cell
Problem: if gene is essential, deletion of it in haploid cells will lead to cell death. It is impossible to study dead things
Solution: Delete the gen in a diploid cell. If cells are diploid, gene replacement will typically only occur on one of the two chromosomes.
Important point: If the gene is essential and we wish to eventually make a homozygous mutant, the diploid cell must also contain a plasmid with the wild-type copy of the gene.
Yeast life cycle
Mating –> Diploid cells (MATa/MATb) –> Sporulation –> Haploid cells (MATa separate from MATb) –> Mating
Meiosis
The result of meiosis is the formation of 4 spores contained together in an ascus (called a tetrad). 2 haploid cells of each
In vitro mutagenesis and plasmid shuffle
In this example, we have taken Your Favorite Gene (YFG) and created a mutation on it (yfg). A yeast with a chromosomal deletion of your favorite gene (yfgΔ ) and a plasmid containing the wild-type copy of YFG is used. Your mutant plasmid is transformed into the cell, so that the cell now contains two plasmids (YFG and yfg).
5-fluororotic acid (5-FOA) can be used to select for cells that lose the wild-type plasmid, because 5-FOA kills cells that contain URA3)
See slide 16
One-step gene replacement to insert mutation
- PCR amplify selectable marker using primers with ends homologous to YFG
- Transform fragment into yeast cells
- Select for cells where SEL has integrated into the chromosome and replaced YFG
Conditional knockout - yeast degron system
See slide 18
Two-step gene replacement
- Transform integrating plasmid containing a mutation of your favorite gene (yfg‐)
- Plasmid containing yfg- recombines into the chromosome as shown in slide 19 (This construct contains both the mutant and wild-type forms of yfg)
- 2nd recombination event occurs between homologous regions of YFG
- If the 2nd recombination event occurs appropriately, the wild-type copy will be recombined out of the chromosome ( an exact replacement of the gene with a mutant copy remains in the chromosome
In vivo cloning
One can utilize homologous recombination in yeast to perform cloning of genes.
PCR can be used to amplify a GOI.
The plasmid is then cleaved with a restriction enzyme to create a ‘gap’ in the vector
The homologous sequences on the PCR fragment and plasmid can be recombined to repair the gap in the plasmid.
The plasmid can then be recovered from the yeast.
This technique can also be used to recover mutated genes in the yeast genome
In vivo cloning - additional uses
More difficult cloning. In Slide 22, a gene is cloned into a gapped vector. In addition, an epitope tag is also being cloned in at the same time. The result will be that when the gene is expressed in cells, the protein produced will be tagged in this case with green fluorescent protein
