MERYL Flashcards
SACCHAROMYCES cerevisae
Single cell yeast
Strip back cell wall - expose to DNA + Ca2+ + PEG (disrupts cell membrane)
Yeast selectable markers
Amino acid auxotrophy
e.g. URA3+ mutant
YIp
Yeast Integrative plasmid Integration by HOMOLOGY LOW transformation frequency MUST integrate to be maintained in yeast Single copy Stable
YEp/YRp
Yeast episomal plasmi/Yeast replicative plasmid = shuttle vectors e..g move DNA between E.coli and yeast YEP - ori: yeast plasmid YRp - ori: yeast chromosome High transformation frequency Not integrated - replicates autonomously in nucleus High copy number Poor segregation during cell division UNSTABLE
YCp
Yeast centromeric plasmid Contain centromeres High transformation Good segregation (centromere) Single copy/cell
YAC
yeast artificial chromsomes Harbour very large pieces of DNA Linear Telomeres (stabilise ends of chromosomes) Not integrated Behave like yeast chromosome
Yeast transformation
Clone by complementation
To find gene: Make genomic library
Defining region of gene involved in regulator expression
- Fuse promoter to reporter gene e.g. lacZ
see where gene is expressed or make deletions until identification of region can be made
Overexpression analysis in yeast
- Use a multicopy plasmid (YRp/YEp) to introduce multiple copies
- Use highly expressed promoter to drive expression of gene
Gene Inactivation & gene replacement
Construct 1:
Insert selectable marker INTO the gene (dbl recom) and DESTROY FUNCTION, select for selectable marker e.g. leu2+ on YIp plasmid
Construct 2:
Insert internal region only of gene into YIp vector
Delete 3’ and 5’ end
SINGLE X-OVER
GENE INACTIVATION
MAKE DIPLOID - keep in good nutritional environment
2 ura3-:2URA3+ = not essential
2 ura3- : 0 URA3+ = essential
Gene replacement
Introduce more subtle mutations in genome e.g. single bp change
