Questions 16.04.2015 Sectoring screen
1. Describe the basis of a colony-color sectoring assay (3)
ade2, ade3double mutant; transformed with plasmid carrying ADE3 gene  red. Can lose plasmid at a
certain rate  plasmid loss cells are white; will appear as sectors in colony. If cells cannot lose plasmid
because it carries an essential gene that is mutated in the cells (cells that lose plasmid will die), colonies
appear red.
2. What is the phenotype of a yeast cell defective in mitochondrial fatty acid synthesis? (1)
Respiratory deficient, loss of cytochromes, lack of lipoic acid, mitochondrial DNA loss
3. What is lipoic acid? (1)
A covalently linked essential cofactor required for the function of several mitochondrial enzyme
complexes. Derivative of octanoic acid with sulfur substituted for hydrogen at carbons 6 and 8.
4. What is a chimera? (1)
(In molecular biology) A fusion of two or more heterologous genes/proteins or domains thereof.
5. Make an outline of the screen for mutations in the yeast mitochondrial 3-hydroxyacyl thioester
dehydratase. (3)
Generation of a chimeric construct consisting of a yeast mitochondrial targeting sequence (COQ3) and a
bacterial 3-hydroxacyl-ACP dehydratase (fabA); the plasmid carries ADE3 as marker (and LEU2).
Transformation into ade2, ade3 mutant strain  colonies are red, as the plasmid is not required, the
colonies lose it even on non-fermentable carbon source  sectored colonies. Mutagenesis of this strain,
screen for colonies that are red (=cannot lose the plasmid) on media containing only non-fermentable
carbon source (control for specificity: colonies DO sector on glucose media)
6. How was the dehydratase cloned? (1)
Transformation of the mutant (containing the plasmid with the chimeric COQ3-fabA construct) with
genomic yeast library. Screening for sectoring or white colonies.
7. How was the ORF encoding -hydroxyacyl-thioester dehydratase on the complementing clone identified?
(1)
Restriction analysis. The library clone harbored convenient restriction sites (HindIII, EcoRI and
KpnI) that allowed to investigate the functional ORF by deletion of parts of the genomic DNA insert.
8. What pieces of evidence supported the hypothesis that YHR067w encodes the 3-hydroxyacyl-thioester
dehydratase of mitochondrial FAS II (3)
The yhr067 mutants are complemented by mitochondrially localized bacterial 3-hydroxyacyl-thioester
dehydratases and suppressed by FAM1-1. Yhr067p has similarity to dehydratases and is localized to
mitochondria. (Overexpression of YHR067w increases hydratase-2 activity in mitochondria
9. How was mitochondrial localization of Yhr067p demonstrated? (1)
Tagging of Yhr067p with protein A (proA); in situ immunofluorescence using FITC-conjugated antibody.
Comparison of fluorescence with the fluorescent mitochondria-specific mitotracker stain
10. How were candidates for the human mitochondrial 3-ketoacyl-ACP reductase identified? (2)
Homology searcher identified proteins with similarity to E. coli fabG and yeast Oar1 ketoreductases.
Candidates predicted to be mitochondrially localized were tested. Initially, complementation of a yeats
oar1 knockout strain failed. After generation of chimeric proteins harboring an N-terminal yeast
mitochondrial targeting sequence, two candidates complemented weakly. Co-transformation of these
candidate proteins gave wild type levels of growth/complementation
11. How was it demonstrated that functional human mitochondrial 3-ketoacyl-ACP reductase was a heteromeric
enzyme consisting of two different components? (2)
Lipoic acid measurements of the oar1strain transformed with the candidates individually or in
combination ruled out a specific function of one candidate in lipoic acid synthesis. Coexpression of the
candidates in E. coli results in heterotetrameric protein complexes. His-tagging of one protein always
results in co-purification of the other protein, even if only one of the proteins is tagged.