Download Review Questions yeast lecture 18

Review Questions yeast lecture 31.01.2017
1. What general minimum requirements does a plasmid have in order to be useful for us as a
tool in molecular biology? (1)
ARS/ori, copy number and plasmid stability information, selectable
marker
2. How can you identify a plasmid that carries a sequence that confers mitotic stability? (2)
Transformation of yeast cells with genomic DNA library. Selection of
transformants that carry the library marker. Pooling of all
transformants and growth over several generations. Mitotically stable
plasmids will enrich in the culture
3. What feature does a yeast plasmid require in order to yield high transformation
frequencies? (1)
ARS (autonomically replicating sequence)
4. Why do plasmids without these features only yield in low transformation frequencies? (1)
Transformation can only occur via homologous recombination, which is
a rare event
5. What type of circular yeast plasmids do you know? What is the principal difference
between them? (1)
YIp, YEp and YCp vectors. Yip do not contain an ARS and have to
integrate to yield transformants. YEp plasmids are present in multiple
copies due to the 2 micron origin of replication. YCp plasmids are
present in low copy number (usually one) due to the centromeric
fragment.
6. What is a YAC? (1)
A yeast artificial chromosome, consisting of centromere ARS, two
markers and telomere fragments. These “Vectors” can be used to clone
an maintain large stretches of DNA
7. Explain the one-step gene replacement technique in yeast (3)
PCR of drug resistance cassette with primers that introduce 40-45bp of
homology to the target gene on each end of the cassette. High efficiency
transformation of yeast cells with the PCR product, selection for drug
resistance. Confirmation of the knockout by PCR, using sets of primers
where one oligo is specific for a sequence within the knockout cassette
or within the target gene, and/or a primer pair that will result in
different size products if the cassette is integrated in the target gene or
not
8. What a plasmid shuffle and in which case is it a required technique? (2)
A plasmid shuffle is a technique where one plasmid in a cell is replaced
by another plasmid via an intermediate that carries two plasmids. This
is a required technique when working with lethal mutations
9. What other technique can you use to introduce a complementing plasmid into a yeast
strain with a lethal mutation? (2)
Transformation into the heterozygous diploid, sporulation of the diploid
and positive/negative selection for the spore colonies containing the
knockout cassette and the plasmid
10. What is a reporter gene? (1)
Reporter genes are heterologous genes that confer and easily detectable phenotype
to the object of study