Review Questions yeast lecture 27.04.2015
1. What is N-linked glycosylation and what role is Rft1p proposed to play in this
process?(2)
Asparagine-linked glycosylation. Rft1p is required to flip a glycan precursor,
synthesized on the cytosol-facing side of the ER, to the ER lumen
2. How was it demonstrated that VLCFA synthesis does not play a role in N-linked
glycosylation (2)
Yeast strains containing mutations in VLCFA synthesis genes and carrying a
plasmids with the corresponding gene under control of the GAL1 promoter were
grown on galactose or glucose, and the glycosylation status of the CPY
(carboxypeptidase Y) protein was examined by Western blot using anti-CPY
antibody. No change of glycosylation was observed upon shutting down expression
of the VLCFA synthesis enzymes.
3. How were multicopy suppressors of the rft1∆ mutation isolated (1)
rft1Δ cells, which can grow on osmostabilized media containing 1 M sorbitol, were
transformed with a multicopy yeast genomic library. Clones were isolated that could
grow on normal media
4. How was it shown that multicopy suppression of the rft1∆ lethal phenotype by
truncated transmembrane proteins is unspecific? (2)
Mutants producing only precursor forms of the flipping substrate were examined in
the rft1Δ strain background for their ability to grow in presence of a plasmid
expressing either RFT1 or a truncated ER membrane protein. Transformants
containing the latter constructs were able to grow even if the glycan synthesis
mutantion only produced a very early precursor, while strains expressing RFT1
only grew if the precursor was still similar to the proper flipping substrate.
5. What is our model for the mode of action of ELO3-H2 in the suppression of the
rft1 mutant phenotype? (2)
Elo3-H2 produces a protein with transmembrane domains that disturb the ER
membrane integrity. A continuum is generated in the membrane surrounding the
Elo3-H2 chimera, allowing for “diffusion” of the glycan precursors into the ER
lumen
6. Describe the basic principle of a DNA microarray use in transcriptional analysis
(1)
Depositing of target DNA (ORFs or oligonucleotides corresponding to ORFs of organism
investigated) onto a solid support . Isolation of RNA of organism under different conditions.
Reverse transcription incorporating fluorescent tag or radioactivity and hybridization of
spotted DNA/oligonucleotide microarray with products from RT. Analysis of
fluorescent/radioactive signal
7. What are the most common formats of DNA “micro” arrays? (1)
Microarray of DNA/cDNA clones on glass slide support, oligonucleotide array on “gene
chip” (affymetrix). “Macroarray” of DNA/cDAN clones on on nylon filter is not common
anymore
8. Describe the advantages, but also the problems of DNA microarray work .(2)
Advantages: Microarray replaces large amounts of northern blots and can be used to
monitor entire transcriptional response of the cells to a challenge. Similar response of genes
may provide clue to function. Pitfalls: limited dynamic range, significance of results needs
to be further established, assumption that genes with related functions are co-regulated is
not always true
9. What are “molecular barcodes” in yeast, and how can they be exploited? (3)
In yeast: Unique sequences that were introduced to the 5’ and 3’ end of the knockout
cassette in all of the knockout strains. These allow identification of specific deletion strains
within a mixture of strains and therefore large scale analysis of effects of selective
conditions (temperature, carbon source, drug,etc.) on individual knockout strains in a
mixed culture.
10. What is “SGA”? Describe the basic principle (3)
Synthetic genetic array. A “Query” strain carrying a non-lethal gene deletion is mated to all
other viable yeast knockout strains. Diploids are sporulated and haploid cells are selected
for using a reporter that is only activated in haploid a- cells. Double deletion mutant is
selected by resistance to two different drugs, and viability of double mutant can be
monitored to determine synthetic lethal interaction.
11. Define the characteristics of “Systems Biology” (2)
Multidisciplinary research with a close interaction between experimental research and
mathematical modeling and analysis. Aspires to generation and iterative improvement of
mathematical models which describe realistically cellular networks or dynamic biological
processes, and which have predictive power. Requires use of existing and generation of
novel data, in particular quantitative data, time courses and spatial information of high
definition