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Flashcards in Lecture 21 Deck (15):

Gene transfer:

- The capacity to inject exogenous DNA into a cell to detect what the function of a gene of interest is.
- In different organisms different systems can be used


Yeast (Saccharomyces cerevisiae):

- Single celled, so can transform quite easily as DNA doesn't need to be inserted into a germ line
- To insert DNA strip back the cell wall so that it becomes osmotically sensitive, in the presence of CA2+ and DNA and PEG (allows DNA to penetrate cell membrane)


Selectable markers used in yeast:

- Usually amino acid auxotrophic markers, eg) URA3+
- Growing the bacteria on a selection plate will only allow ura3-. Recipients of the DNA of interest will be the only ones growing



- Yeast integrative plasmid
- Must integrate into the yeast genome to be maintained
- Integration occurs through homology or a single cross over event
- This has a low transformation frequency
- Integrated, usually single copy and stable


Adding a yeast Origin of repliction:

- More transformants will be generated
- Integration step is a limiting step for YIP



- The difference is in where the origin of replication comes from
- YEp: Yeast episomal plasmid, OriC from a yeast plasmid
- YRp: Yeast replicated plasmid from a yeast chromosome, OriC from yeast chromosome
- Transform yeasts at high frequency, not integrate and replicate autonomously within the nuclues
- Not integrated, high copy number, unstable


YEp/YRp uses:

- Shuttle vectors for carrying DNA between E.coli and yeast for example
- Replicates in E.coli as it has an E.coli OriC and an E.coli selectable marker
in addition to having a yeast OriC and selectable marker
- Replicates in both organisms so is very useful



- Yeast Centromeric plasmid
- Similar to YEp/YRp, but also have a centromere
- It was have all the features of the shuttle vector, but the centromere also means that the plasmids divide correctly after cell division and are maintained in single copy
- Good segregation and high transformation frequency
- Not integrated, single copy, stable



- Yeast Artificial chromosome
- Used to harbour large amounts of DNA with great stability
- Telomeres at the ends means that the DNA isn't degraded and the YAC's are stabilised
- Not integrated, behaves like a yeast chromosome


Genes can be cloned by complementation:

- A mutant with an identifiable phenotype.. clone the gene via complementation
- Take the gene and introduce a plasmid containing a WT copy of the gene, so the mutant can regain a WT phenotype
- This implies that we don't know what the gene is to start off with
- So to figure out what gene it is, we can create a whole genomic library


Sources of WT genes:

- Cut vector and cut genomic DNA can be ligated to form a genomic library shuttle vector
- We can then transform the library into the yeast mutant


Do you need to know if your mutation is dominant or recessive?

- Yes!
- If the mutant phenotype is dominant what do we do?
- Answers in next lecture


Using transformation in yeast to investigate gene expression using reporter genes:

- Take promoter of interest and fuse it to a reporter gene (eg, lacZ gene of E.coli)
- Introduce the lacZ gene and the promoter of interest and introduce into yeast, lacZ will report when your promoter is active
- Can make deletions until you identify the regions bringing about regulation


Other uses of Yeast transformation:

- Investigating gene expression using reporter genes
- Over expression analysis and regulation of gene expression
- Gene inactivation and gene replacement
- Cloning by complementation


Over expression analysis:

- Replace the promoter of interest with a strong promoter that will drive over expression of the gene of interest
- Add a multicopy plasmid (YRp/YEp) to introduce multiple copies of your gene