Lecture 21

Question 1

Gene transfer:

Answer 1

• 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

Question 2

Yeast (Saccharomyces cerevisiae):

Answer 2

• 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)

Question 3

Selectable markers used in yeast:

Answer 3

• 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

Question 4

YIP:

Answer 4

• 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

Question 5

Adding a yeast Origin of repliction:

Answer 5

• More transformants will be generated

- Integration step is a limiting step for YIP

Question 6

YEp/YRp:

Answer 6

• 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

Question 7

YEp/YRp uses:

Answer 7

• 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

Question 8

YCp:

Answer 8

• 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

Question 9

YAC:

Answer 9

• 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

Question 10

Genes can be cloned by complementation:

Answer 10

• 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

Question 11

Sources of WT genes:

Answer 11

• 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

Question 12

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

Answer 12

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

Question 13

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

Answer 13

• 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

Question 14

Other uses of Yeast transformation:

Answer 14

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

Question 15

Over expression analysis:

Answer 15

• 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