= Yeast cross-species complementation experiment
Can a <gene> from <Schizosaccharomyces pombe> replace a missing function in <Saccharomyces cerevisiae>? A modern laboratory project tested <functional complementation> of <methionine> synthesis. For each tested <gene deletion>, the investigators compared <plasmids> carrying the corresponding <Saccharomyces cerevisiae> <gene>, a candidate <Schizosaccharomyces pombe> <gene>, and an unrelated <gene>. The first was a positive <scientific control>; the last was a negative <scientific control>. The paper describes this design rather than publishing a complete set of individual growth measurements. <Pathways over Time: Functional Genomics Research in an Introductory Laboratory Course by Todd D. Reeves and others>[Reeves, Warner, Ludlow and O’Connor (2018), Course Activities, pp. 5–6 and Table 1: three plasmids, replica plating and protein-expression checks]{ref}
The recipient <biological strains> also lacked the ability to make <uracil>. The pYES2.1/V5-His <plasmids> carried URA3, a <gene> restoring that ability, and used the GAL1 <gene promoter> to control the inserted <gene>. Initial plates lacked <uracil> but supplied <methionine>: this selected <biological colonies> containing <plasmids> without requiring successful <methionine> synthesis. Subsequent plates lacking <methionine> tested the missing function, comparing <galactose induction> with <glucose> conditions. Thus selecting for a <plasmid> and testing its functional contribution were separate operations. <Pathways Over Time Plasmids Engage Students in Functional Genomics Research by Clare O’Connor>[O’Connor (2018), Plasmid features enable multiple student experiments and Complementation reveals conservation of function]{ref}
The apparatus can be simple and informative. In the associated protocol, <replica plating> uses a block covered with velveteen to transfer small samples of <biological colonies> from a master plate onto plates with different nutrient conditions. Orientation marks preserve correspondence between locations; gentle contact limits the transferred material. The specified replicas omit <uracil> and <methionine> with either inducing <sugar> or <glucose>, and include a fresh <glucose> plate supplying <methionine>. The readout is growth at corresponding locations, not a direct measurement of <enzyme> activity. <Investigations in Molecular Cell Biology by Clare M. O’Connor/12.6: Exercise 2 - Replica plating and complementation>[O’Connor, Investigations in Molecular Cell Biology, §12.6, introductory paragraphs and steps 1–3: velveteen block, orientation marks and three replica media]{ref}
\Image[/yeast-gene-complementation-experimental-design.png]
{title=Separating <plasmid> selection from a <yeast> <functional complementation> test}
{description=Original schematic of the reported experimental design, not copied growth data. All three constructs enter the same <Saccharomyces cerevisiae> <gene deletion> background. Plate labels identify distinct comparisons; arrows show sample transfer, not observed rescue. The candidate <Schizosaccharomyces pombe> outcome must be measured.}
The <scientific controls> answer different objections. Growth on the <methionine>-supplied replica checks survival and transfer. Rescue by the native <gene> checks that this <biological strain> and assay can reveal <functional complementation>. The unrelated <gene> tests whether carrying a <plasmid> alone is sufficient. Comparing induction conditions checks dependence on <gene expression>, although changing the <sugar> also changes <metabolism>: it is not an isolated change of <gene expression> in every other respect.
If the candidate <gene> restores growth while those comparisons behave as intended, it supplies a function sufficient for the tested growth requirement in this recipient. Failure to rescue is less decisive: the candidate <protein> might not be produced, might be altered by its attached tag, or might not work in this cellular context. The investigators therefore also used <Western blots> to examine <protein> production. A detected <protein> still does not establish correct <protein folding> or catalytic activity. <Pathways over Time: Functional Genomics Research in an Introductory Laboratory Course by Todd D. Reeves and others>[Reeves et al. (2018), Course Activities, pp. 5–6: functional conservation, tag interference and Western-blot follow-up]{ref}
Back to article page
